Dehydroepiandrosterone modulates antioxidant enzymes and Akt signaling in healthy Wistar rat hearts

Dehydroepiandrosterone (DHEA) is an endogenous steroid synthesized mainly in the adrenal cortex. It is known that DHEA is a precursor of sex steroids and that part of its effects depends on its conversion to estrogens and androgens. Sex steroids exert profound and controversial effects on cardiovascular function. Exogenous DHEA can exert a dual effect, antioxidant or prooxidant, depending on the dose and on the tissue specificity [1,2] (F. Celebi, I. Yilmaz, H. Aksoy, M. Gümüs, S. Taysi, D. Oren, Dehydroepiandrosterone prevents oxidative injury in obstructive jaundice in rats, J. Int. Med. Res. 32 (4) (2004) 400-405; S.K. Kim, R.F. Novak, The role of intracellular signaling in insulin-mediated regulation of drug metabolizing enzyme gene and protein expression, Pharmacol. Ther. 113 (1) (2007) 88-120). Akt signaling pathway is one of the anti-proliferative mechanisms of DHEA (Y. Jiang, T. Miyazaki, A. Honda, T. Hirayama, S. Yoshida, N. Tanaka, Y. Matsuzaki, Apoptosis and inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway in the anti-proliferative actions of dehydroepiandrosterone, J. Gastroenterol. 40 (5) (2005) 490-497). Heart homogenates were prepared to quantify lipid peroxidation (LPO), concentration of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), 4-hydroxy-2-nonenal (HNE) and p-Akt/Akt ratio, and the activities of those antioxidant enzymes. When administrated to male Wistar rats in short-term (6 or 24h) intraperitoneally, DHEA produced significant differences in some parameters of oxidative stress in rat hearts among the distinct doses (1, 10, and 50mg/kg) used. The results here presented, regarding 6 and 24h oxidative stress status, have shown that DHEA injections promoted a prooxidant answer in healthy Wistar rat hearts.     

Thyrotropin-releasing hormone stimulates intestinal transit in young rats

The effect of intracisternal injection of thyrotropin-releasing hormone (TRH) on small intestinal transit of a charcoal bolus was investigated in 14-, 21-, 28- and 35-day-old and adult rats. Intracisternal TRH (15 micrograms in 2 microliters) was administered, and transit (distance traveled by the charcoal) was measured 120 min later. In all age groups, intracisternal TRH increased charcoal transit significantly (P less than 0.05) as compared to saline-treated controls. This increase in transit was not mimicked by intravascular TRH, and it was blocked in all age groups by prior intraperitoneal injection of atropine (2 micrograms/g body weight). Vagotomy blocked TRH-induced increases in small intestine transit in rats of 28 days and older. Prior intraperitoneal injection of the antiserotonin compound, cyproheptadine (1 microgram/g body weight) reduced TRH-induced increases in small intestine transit in all age groups. These results demonstrate that centrally administered TRH stimulates small intestine transit through both cholinergic and serotonergic mechanisms in rats as early as 14 days of age.     

Regeneration and transplantation of muscles in old rats and between young and old rats.

In order to compare the regenerative ability of skeletal muscle between young (5 month) and old (26 month) rats, sliced or intact extensor digitorum longus muscles were freely autografted into young and old rats and also reciprocally grafted from young to old inbred animals and vice versa. Sixty days after grafting, the transplants were analyzed for contractile and histochemical properties. There was a relative similarity between the contraction times of both normal control muscles and of all groups of transplants, although the contraction time tended to be prolonged and histochemical fiber pattern was more often found to be uniform in grafts of senescent animals. All groups of transplants possessed histochemically heterogeneous fiber types at 60 days. The experiments demonstrate that skeletal muscle in old rats possesses a substantial degree of regenerative ability and that the free tranpllantation of entire muscles in old animals is feasible.     

Fever and behavioral thermoregulation in young and old rats

At standard laboratory ambient temperatures (T(a)) of 20-24 degrees C, peripheral injections of lipopolysaccharide (LPS) reliably produce fever in young rats. In contrast, old rats may show a blunted fever, no fever, or even hypothermia after LPS. In the present study we hypothesized that old rats might use behavioral thermoregulation to help them develop a fever. Young and old rats were implanted with temperature transmitters. At least 1 wk postoperatively they were placed in a thermally graded alleyway (T(a) 10-40 degrees C). On the third and sixth day they were taken out of the gradient, placed at an T(a) of 23 degrees C, injected intraperitoneally with LPS or saline, and left at 23 degrees C for 3 h. At the end of that time, all young rats had become febrile, whereas the old rats had not. When the rats were replaced in the thermal gradient, the young animals continued to develop a fever that was similar to fever in young rats left at 23 degrees C. The old animals chose significantly warmer positions in the thermal gradient than did the young animals and only then became febrile. Although there was a tendency for the young rats to prefer higher T(a) after LPS than after saline, these differences were not significant. However, the differences in the old rats were significant. These results suggest that the LPS had increased the thermal set point in the old rats, but they could develop febrile responses only at the warm T(a) they selected.     

The MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signaling

The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer''s disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo. We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices or primary neurons treated with an MT2 receptor selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3β/CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3β/CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons.Synaptic circuits are established at the sites of axon–dendritic, axon–somatic or axon–axonal contact, in which functional axonogenesis is a critical step.¹ Axonogenesis can be regulated by many intracellular signals that involve cytoskeletal rearrangements,² local protein degradation,³ as well as diffusional barriers.⁴ Additionally, several extracellular neurotrophic factors and hormones have also been shown to have a role in axon guidance and synaptic formation in central neurons.^{5, 6} To date, the role of melatonin and its receptors in axonogenesis remains unclear. Most of the biological functions of melatonin are mediated by its two receptors, MT1 and MT2 receptors, both of them belong to the G protein-coupled receptor (GPCR) subfamily and are widely expressed throughout the central nervous system (CNS).⁷ Activation of the MT2 receptor in response to melatonin is critical for controlling circadian rhythms⁷ and regulation of slow wave sleep.^{8, 9} Early studies have shown that activation of the MT2 receptor in the retina reduces the release of dopamine, while dopamine inhibits growth cone motility and neurite outgrowth during embryonic development,^{10, 11} suggesting the involvement of the MT2 receptor in functional axonogenesis. In mutant mice with deficient expression of the MT2 gene, the induction of long-term potentiation (LTP) of excitatory synaptic transmission is impaired, and this impairment is closely related to deficits in learning.¹² In the hippocampus, the MT2 receptor inhibits GABA_A receptor-mediated current,¹³ which is implicated in the synaptic transmission. In Alzheimer''s disease, expression of the MT2 receptor is significantly reduced, especially in the hippocampus.^{12, 14, 15} A partial agonist of the MT2 receptor, UCM765, exhibits anxiolytic-like properties by increasing the time spent in the open arm of an elevated plus maze test, and by reducing the latency to eat in a novel environment in the novelty suppressed feeding test, suggesting its role in anxiety.¹⁶ Together, these findings suggest that the MT2 receptor links the signaling cascades that mediate learning and memory formation, one of the important biological functions of melatonin;¹⁷ however, the cellular and molecular events underlying this linkage are yet to be established.Dissociated hippocampal neurons have been commonly used as an excellent in vitro model in the study of axon development and synaptic transmission because they maintain morphological, functional and molecular characteristics of the hippocampal neurons in vivo.¹⁸ In dissociated hippocampal neurons, the transition for axon formation and maturation involves the following five stages:¹⁹ stage 1 neurons (~2 to 4 h after plating) display abundant lamellipodia and filopodia that develop into several immature short neurites at stage 2 (~12 to 24 h); polarization occurs at stage 3 (~24 to 48 h), in which a single neurite initiates a rapid elongation to become the axon while others acquire dendritic identity; stage 4 (~3–4 days) is characterized by rapid outgrowth of axon and dendrites; and at stage 5 (7 days onwards), the maturation of axon and dendrites is essential for functional synapse formation.^{20, 21} In the present study, we have identified a novel role for the MT2 receptor in functional axonogenesis and show that activation of the MT2 receptor is crucial for functional axonogenesis and synaptic transmission in central neurons. Using fluorescence resonance energy transfer (FRET) imaging combined with peptide blocking assays, we have identified Akt as an interacting partner and a substrate of the MT2 receptor. Activation of the MT2 receptor-Akt signaling cascade promotes the formation of functional synapses in the hippocampus, whereas inhibition of the MT2 receptor arrests axonogenesis and synaptic transmission. Given the implications of the MT2 receptor in learning and memory, we propose that targeting MT2 receptor-Akt signaling may be a feasible strategy for stimulating functional synaptic circuit assembly.     

Phosphatidylinositol 3-kinase/Akt signaling stimulates colonic mucosal cell survival during aging

Although aging is shown to be associated with decreased apoptosis and increased survival of cells in the colonic mucosa of Fischer 344 rats, the regulatory mechanisms are poorly understood. The current investigation examines the involvement of phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway in mediating the events of colonic mucosal cell survival during aging. We have observed that aging is associated with activation of PI3K/Akt signaling, as evidenced by the higher levels of phosphorylated forms of p85, the regulatory subunit of PI3K and of Akt in the proximal and distal colonic mucosa, of aged (21-23 mo) than in young (4-7 mo) rats. These increases are accompanied by a concomitant rise in phosphorylation of proapoptotic protein Bad, which is sequestered by the 14-3-3 family of proteins following phosphorylation by Akt, resulting in a reduction in nonphosphorylated Bad. The amount of antiapoptotic Bcl-xL bound to nonphosporylated Bad in the colonic mucosa is found to be substantially lower in aged than in young rats, resulting in a marked rise in the unbound/free form of Bcl-xL in the aging colon. The age-related activation of PI3K and the reduction in caspase-3 activity could be reversed by wortmannin, a specific inhibitor of PI3K. Increased levels of Bcl-xL and phosphorylated forms of Akt and Bad and reduction in caspase-3 activity were observed throughout the entire length of the colonic crypt of aged rats. We conclude that the constitutive activation of the PI3K/Akt-signaling pathway is partly responsible for the age-related increase in colonic mucosal cell survival. This is evident throughout the entire length of the colonic crypt.     

Melatonin protects against methotrexate hepatotoxicity in young rats: Impact of PI3K/Akt/mTOR signaling

With the improvement in children's acute lymphoblastic leukemia (ALL) care, the survival rate in children ALL has improved much. Methotrexate (MTX) plays an essential role in the success of children's ALL treatment. Since hepatotoxicity is commonly reported in individuals treated with intravenous or oral MTX, our study further examined the hepatic effect following intrathecal MTX treatment, which is an essential treatment for leukemia patients. Specifically, we examined the pathogenesis of MTX hepatotoxicity in young rats and explored the impact of melatonin treatment in protection against MTX hepatotoxicity. Successfully, we found that melatonin was able to protect against MTX hepatotoxicity.     

Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells

Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein kinase B, and endothelial nitric oxide synthesis (eNOS) in HUVECs. Transduction with either dominant-negative AMPK or dominant-negative Akt abolished adiponectin-induced eNOS phosphorylation as well as adiponectin-stimulated HUVEC migration and differentiation. Dominant-negative AMPK also inhibited adiponectin-induced Akt phosphorylation, suggesting that AMPK is upstream of Akt. Dominant-negative Akt or the phosphatidylinositol 3-kinase inhibitor LY294002 blocked adiponectin-stimulated Akt and eNOS phosphorylation, migration, and differentiation without altering AMPK phosphorylation. Finally, adiponectin stimulated blood vessel growth in vivo in mouse Matrigel plug implantation and rabbit corneal models of angiogenesis. These data indicate that adiponectin can function to stimulate the new blood vessel growth by promoting cross-talk between AMP-activated protein kinase and Akt signaling within endothelial cells.     

Transient hypobaric hypoxia improves spatial orientation in young rats

To achieve a better understanding of learning and declarative memory under mild transient stress, we investigated the effect of brief hypobaric hypoxia on spatial orientation in rats. Young male Wistar rats aged 30 days were exposed for 60 min to hypobaric hypoxia, simulating an altitude of 7,000 m (23,000 ft) either shortly prior to attempting or after mastering an allothetic navigation task in the Morris water maze with a submerged platform. The post-hypoxic group performed significantly better in the navigation task than the control animals (the mean difference in escape latencies was 11 seconds; P=0.0033, two-way ANOVA with repeated measures, group x session). The experimental group also achieved a remarkably higher search efficiency (calculated as a percentage of successful trials per session), especially during the first four days following hypoxic stress (P=0.0018). During the subsequent training, the post-hypoxic group performed better than the control animals, whilst the efficiency levels of both groups progressively converged. Spatial memory retention and recall of well-trained rats were not affected by the transient hypobaric hypoxia. These results indicate that brief hypobaric hypoxia enhances rats' spatial orientation. Our findings are consistent with several studies, which also suggested that mild transient stress improves learning.     

Dehydroepiandrosterone increases beta-cell mass and improves the glucose-induced insulin secretion by pancreatic islets from aged rats

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose-induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt-1, and Akt-2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA-treated rats showed an increased beta-cell mass accompanied by increased Akt-1 protein level but reduced IR, IRS-1, and IRS-2 protein levels and enhanced glucose-stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.     

Thermoregulatory and metabolic changes during fever in young and old rats

We injected old and young rats with lipopolysaccharide (LPS; 50 microg/kg ip) at two ambient temperatures (Ta; 21 and 31 degrees C). Young rats mounted equivalent fevers at both Tas [peak body temperatures (Tb) of 38.3 and 38.7 degrees C, respectively]. The Tb of old rats was not different from baseline (37.3 degrees C) after LPS at Ta 21 degrees C, whereas, at 31 degrees C, their Tb rose to a mean peak of 38.4 degrees C. We also measured the associated thermoregulatory responses by use of calorimetry. At 21 degrees C, young rats developed a fever by increasing both O2 consumption and heat conservation. Old rats did not become febrile, and O2 consumption fell by 15%. Heat loss was the same in old and young rats. At 31 degrees C, young and old rats developed similar fevers with similar increases in heat production and conservation. Our results suggest that the lack of LPS fever in old rats at 21 degrees C is due mainly to the lowered metabolic rate.     

Trehalose supplementation reduces hepatic endoplasmic reticulum stress and inflammatory signaling in old mice

The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging and cellular damage. Quality control systems, such as the unfolded protein response (UPR), inflammatory signaling and protein degradation, mitigate the residence time of damaged proteins. In the present study, we have examined the UPR and inflammatory signaling in the liver of young (~6 months) and old (~28 months) mice (n=8/group), and the ability of trehalose, a compound linked to increased protein stability and autophagy, to counteract age-induced effects on these systems. When used, trehalose was provided for 4 weeks in the drinking water immediately prior to sacrifice (n=7/group). Livers from old mice were characterized by activation of the UPR, increased inflammatory signaling and indices of liver injury. Trehalose treatment reduced the activation of the UPR and inflammatory signaling, and reduced liver injury. Reductions in proteins involved in autophagy and proteasome activity observed in old mice were restored following trehalose treatment. The autophagy marker, LC3B-II, was increased in old mice treated with trehalose. Metabolomics analyses demonstrated that reductions in hexosamine biosynthetic pathway metabolites and nicotinamide in old mice were restored following trehalose treatment. Trehalose appears to be an effective intervention to reduce age-associated liver injury and mitigate the need for activation of quality control systems that respond to disruption of proteostasis.     

Bone mechanical properties after exercise training in young and old rats

The effects of a 10-wk training regimen on the mechanical properties of the femur and humerus were evaluated in 2.5- and 25-mo-old Fischer 344 female rats. The rats trained on a rodent treadmill 5 days/wk for 10 wk. Duration, grade, and speed increased until the rats maintained 1 h/day at 15% grade and either 15 m/min (old rats) or 36 m/min (young rats). Excised bones were mechanically tested with a 3-point flexure test for mechanical properties of force, stress, and strain. Fat-free dry weight (FFW) and moment of inertia were also obtained. With aging, similar increases were observed in both the femur and humerus for FFW, moment of inertia, and force. Ultimate stress was reduced in the senescent femur while strain was elevated; a similar but nonsignificant trend was observed in the humerus. Irrespective of age, training increased FFW in the femur and, to a lesser degree, in the humerus. Breaking force was elevated for both bones after training. In young and old bones, the training-induced differences in bone mass and force were similar, despite differences in training intensity. In the old trained rats, femur ultimate stress was greater than that in control rat femurs and similar to that in young rat femurs. The results of the present study indicate that training effects were not limited by age.