Energy-regulated molecules maintain young status in the trophocytes and fat cells of old queen honeybees

Queen honeybees (Apis mellifera) have much longer lifespans than worker bees. Energy-regulated molecules in the trophocytes and fat cells of workers during aging have been determined, but are unknown in queen bees. In the present study, energy-regulated molecules were evaluated in the trophocytes and fat cells of young and old queen bees. Adenosine monophosphate-activated protein kinase α2 (AMPK-α2), phosphorylated AMPK-α2 (pAMPK-α2), and cAMP-specific phosphodiesterases activity increased with aging. The pAMPK-α2/AMPK-α2 ratio and AMPK activity; adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) concentrations; the ADP/ATP ratio and the AMP/ATP ratio; the cyclic adenosine monophosphate concentration; forkhead box protein O expression; Silent information regulator T1 (SirT1) expression and activity; and peroxisome proliferator-activated receptor-α (PPAR-α) expression were not significantly different between young and old queen bees. These results show that energy-regulated molecules maintain a youthful status in the trophocytes and fat cells of queen bees during aging. These cells seem to have longevity-promoting mechanisms and may clarify the secret of longevity in queen bees.     

Autophagy in the heart and liver during normal aging and calorie restriction

Autophagy is a highly regulated intracellular process for the degradation of cellular constituents and essential for the maintenance of a healthy cell. We evaluated the effects of age and life-long calorie restriction on autophagy in heart and liver of young (6 months) and old (26 months) Fisher 344 rats. We observed that the occurrence of autophagic vacuoles was higher in heart than liver. The occurrence of autophagic vacuoles was not affected by age in either tissue, but was increased with calorie restriction in heart but not in liver. Next, we examined the expression of proteins involved in the formation and maturation of autophagosomes (beclin-1, LC3, Atg7, Atg9) or associated with autolysosomes and lysosomes (LAMP-1; cathepsin D). In hearts of both ad libitum-fed and calorie-restricted rats, we observed an increase in expression of beclin-1 and procathepsin D, but not mature cathepsin D, and a decrease in expression of LAMP-1 because of aging. In hearts, calorie restriction stimulated the expression of Atg7 and Atg9 and the lipidation of Atg8 (elevated LC3-II/I ratios) in aged rats. In hearts of ad libitum-fed rats, expression of Atg7 and lipidation of Atg8 were unaffected by age, while the cellular levels of Atg9 were lower in aged animals. Furthermore, we observed that the age- and diet-dependent expression levels of those proteins differed between heart and liver. In conclusion, autophagy in heart and liver did not decrease with age in ad libitum-fed rats, but was enhanced by calorie restriction in the heart. Thus, calorie restriction may mediate some of its beneficial effects by stimulating autophagy in the heart, indicating the potential for cardioprotective therapies.     

Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity

In most animals, longevity is achieved at the expense of fertility, but queen honey bees do not show this tradeoff. Queens are both long-lived and fertile, whereas workers, derived from the same genome, are both relatively short-lived and normally sterile. It has been suggested, on the basis of results from workers, that vitellogenin (Vg), best known as a yolk protein synthesized in the abdominal fat body, acts as an antioxidant to promote longevity in queen bees. We explored this hypothesis, as well as related roles of insulin-IGF-1 signaling and juvenile hormone. Vg was expressed in thorax and head fat body cells in an age-dependent manner, with old queens showing much higher expression than workers. In contrast, Vg expression in worker head was much lower. Queens also were more resistant to oxidative stress than workers. These results support the hypothesis that caste-specific differences in Vg expression are involved in queen longevity. Consistent with predictions from Drosophila, old queens had lower head expression of insulin-like peptide and its putative receptors than did old workers. Juvenile hormone affected the expression of Vg and insulin-IGF-1 signaling genes in opposite directions. These results suggest that conserved and species-specific mechanisms interact to regulate queen bee longevity without sacrificing fecundity.     

Honeybee trophocytes and fat cells as target cells for cellular senescence studies

Trophocytes and fat cells are distributed around the abdominal segments in honeybees (Apis mellifera). Whether these cells are a good model for cellular senescence studies is unknown. Here we used histochemical, biochemical, and genetic techniques to investigate the fluctuation of age-related molecules in trophocytes and fat cells of newly emerged and old worker bees. Histochemical studies revealed that old worker trophocytes and fat cells exhibited more senescence-associated β-galactosidase, lipofuscin granules, and non-homogeneous cellular morphology compared to the same cells in newly emerged workers. Biochemical assays demonstrated that trophocytes and fat cells of old workers expressed more lipid peroxidation and protein oxidation than those of newly emerged workers. Molecular genetic analyses detected no difference in telomerase activity or telomere length in trophocytes and fat cells between newly emerged and old workers, showing that these cells do not divide in adulthood. These results suggest that the expression of age-related molecules in trophocytes and fat cells is associated with the senescence of the cells and that honeybee trophocytes and fat cells can serve as a mode for cellular senescence.     

Association of increased autophagic inclusions labeled for beta-galactosidase with fibroblastic aging

Replicative senescence appears after a finite number of cell divisions. After proliferation has ceased, senescent cells remain viable for long periods and metabolic modifications are observed such as lipofuscin accumulation. In order to understand this phenomenon, we examined the emergence of subcellular modifications corresponding to autophagy in MRC5 normal human fibroblasts. An increase of monodansylcadaverine fluorescence, a specific marker of autophagy, in aging compared to young fibroblasts was observed (p<0.0001). The increase of autophagic vacuoles in aging fibroblasts was confirmed by electron microscopy. We compared young versus senescent fibroblasts and showed that autophagic vacuoles, already present in young cells, became larger in senescent fibroblasts with a significant relative increase of inclusion area with respect to measured cell area (p=0.0041). However, autophagy-associated-gene expression remained stable in senescent compared to young fibroblasts, suggesting that the autophagy process per se is not enhanced. In parallel, transmission electron microscopy analysis showed that beta-galactosidase activity distribution was modified by aging: beta-galactosidase (an enzyme linked to lysosome) was scattered in young fibroblasts, but clustered at the level of autophagic vacuoles in senescent fibroblasts, suggesting a predominance of autolysosomes at this stage. These results support the hypothesis that, during fibroblast aging, the increase of autophagic vacuoles, as well as that of beta-galactosidase activity, may be associated to an increase of lysosomal mass and to an accumulation of degradative autolysosomes with lipofuscin. This phenomenon could be involved in the death of senescent fibroblasts.     

Age-related autophagy alterations in the brain of senescence accelerated mouse prone 8 (SAMP8) mice

Autophagy is responsible for the degradation of long-lived proteins and damaged organelles intracellular, even extracellular,and autophagy is proved to have relationship with Alzheimer's disease (AD) and aging. The senescence accelerated mouse prone 8 (SAMP8) was a non-genetically modified mice widely used as a rodent model of aging and senile dementia. However, little was known about the age-related changes of autophagy in the brain of SAMP8 mice. To better understand the precise relationship between aging, autophagy and neurodegeneration, we explored the time course of cognitive ability, ubiquitin-positive inclusions, ultrastructure of neurons and detected the expression of LC3 and Beclin 1 protein in different brain regions of 2, 7 and 12-month-old SAMP8 and SAMR1 mice. We found that 7 and 12-month-old SAMP8 mice presented cognitive decline and ubiquitinated proteins enhanced. In the hippocampal neurons of 12-month-old SAMP8 mice, lots of dense clumps and autophagic vacuoles were found in the cytoplasm and axons. The LC3-II expression showed an increase in hippocampus and cortex of 7 and 12-month-old SAMP8 mice. The expression of Beclin 1 displayed a significant increase in 7 months old and a decline in 12 months old mice. Based on these data, we suggest that the autophagic activity maybe increase reactively at the beginning of AD and then showed a decline with aging, and the pathological changes of 12-month-old SAMP8 mice are more similar to the late-onset AD in the perspective of autophagy.     

Changes in mitochondrial energy utilization in young and old worker honeybees (Apis mellifera)

Trophocytes and fat cells in honeybees (Apis mellifera) have served as targets for cellular senescence studies, but mitochondrial energy utilization with advancing age in workers is unknown. In this study, mitochondrial energy utilization was evaluated in the trophocytes and fat cells of young and old workers reared in a field hive. The results showed that (1) mitochondrial density increased with advancing age; (2) mitochondrial membrane potential (∆Ψm), nicotinamide adenine dinucleotide oxidized form (NAD⁺) concentration, adenosine triphosphate (ATP) concentration, and NAD⁺/nicotinamide adenine dinucleotide reduced form (NADH) ratio decreased with advancing age; and (3) the expression of NADH dehydrogenase 1 (ND1), ATP synthase, and voltage-dependent anion channel 1 (VDAC1) increased with advancing age, whereas ND1 and ATP synthase did not differ with advancing age after normalization to mitochondrial density and VDAC1. These results show that the trophocytes and fat cells of young workers have higher mitochondrial energy utilization efficiency than those of old workers and that aging results in a decline in mitochondrial energy utilization in the trophocytes and fat cells of worker honeybees.     

Ambient temperature reduction extends lifespan via activating cellular degradation activity in an annual fish (Nothobranchius rachovii)

Ambient temperature reduction (ATR) can extend the lifespan of organisms, but the underlying mechanism is poorly understood. In this study, cellular degradation activity was evaluated in the muscle of an annual fish (Nothobranchius rachovii) reared under high (30 °C), moderate (25 °C), and low (20 °C) ambient temperatures. The results showed the following: (i) the activity of the 20S proteasome and the expression of polyubiquitin aggregates increased with ATR, whereas 20S proteasome expression did not change; (ii) the expression of microtubule-associated protein 1 light chain 3-II (LC3-II) increased with ATR; (iii) the expression of lysosome-associated membrane protein type 2a (Lamp 2a) increased with ATR, whereas the expression of the 70-kD heat shock cognate protein (Hsc 70) decreased with ATR; (iv) lysosome activity increased with ATR, whereas the expression of lysosome-associated membrane protein type 1 (Lamp 1) did not change with ATR; and (v) the expression of molecular target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR) as well as the p-mTOR/mTOR ratio did not change with ATR. These findings indicate that ATR activates cellular degradation activity, constituting part of the mechanism underlying the longevity-promoting effects of ATR in N. rachovii.     

Oxidative stress decreases in the trophocytes and fat cells of worker honeybees during aging

Trophocytes and fat cells of honeybees have been used for cellular senescence studies, but their oxidative stress and antioxidant enzyme activities with aging in workers is unknown. Here, we assayed reactive oxygen species and the activities of antioxidant enzymes in the trophocytes and fat cells of young and old workers. Young workers had higher reactive oxygen species levels, higher superoxide dismutase and thioredoxin reductase activities as well as lower catalase and glutathione peroxidase activities compared to old workers. Adding these results up, we propose that oxidative stress decreases with aging in the trophocytes and fat cells of workers.     

Low-level caloric restriction rescues proteasome activity and Hsc70 level in liver of aged rats

Proteasome activity is known to decrease with aging in ad libitum (AL) fed rats. Severe caloric restriction (CR) significantly extends the maximum life-span of rats, and counteracts the age-associated decrease in liver proteasome activities. Since few investigations have explored whether lower CR diets might positively counteract the age associated decrease in proteasome activity, we then investigated the effects of a mild CR regimen on animal life-span, proteasome content and function. In addition, we addressed the question whether both CR regimens might also affect the expression of Hsc70 protein, a constitutive chaperone reported to share a role in the function of proteasome complex and in the repair of proteotoxic damage, and whose level decreased during aging. In contrast to severe CR, mild CR had a poor effect on life-span; however, it better counteracted the decrease of proteasome activities. Both regimens, however, maintain Hsc70 in liver of old rats at level comparable to that of young rats. Interestingly, the effects of aging and CRs on liver proteasome enzyme activities did not appear to be associated with parallel changes in the amount of proteasome proteins suggesting that the quality (molecular activity of the enzymes) rather than the quantity are likely to be modified with age. In conclusion, the results presented in this work show that a mild CR can have beneficial effects on liver function of aging rats because is adequate to counteract the decrease of proteasome function and Hsc70 chaperone level.     

The use of honeybees reared in a thermostatic chamber for aging studies

Honeybees (Apis mellifera) are an attractive model system for studying aging. However, the aging level of worker honeybees from the field hive is in dispute. To eliminate the influence of task performance and confirm the relationship between chronological age and aging, we reared newly emerged workers in a thermostat at 34°C throughout their lives. A survivorship curve was obtained, indicating that workers can be reared away from the field hive, and the only difference between these workers is age. To confirm that these workers can be used for aging studies, we assayed age-related molecules in the trophocytes and fat cells of young and old workers. Old workers expressed more senescence-associated β-galactosidase, lipofuscin granules, lipid peroxidation, and protein oxidation than young workers. Furthermore, cellular energy metabolism molecules were also assayed. Old workers exhibited less ATP concentration, β-oxidation, and microtubule-associated protein light chain 3 (LC3) than young workers. These results demonstrate that honeybees reared in a thermostatic chamber can be used for aging studies and cellular energy metabolism in the trophocytes and fat cells of workers changes with advancing age.     

Queen pheromone modulates brain dopamine function in worker honey bees

Honey bee queens produce a sophisticated array of chemical signals (pheromones) that influence both the behavior and physiology of their nest mates. Most striking are the effects of queen mandibular pheromone (QMP), a chemical blend that induces young workers to feed and groom the queen and primes bees to perform colony-related tasks. But how does this pheromone operate at the cellular level? This study reveals that QMP has profound effects on dopamine pathways in the brain, pathways that play a central role in behavioral regulation and motor control. In young worker bees, dopamine levels, levels of dopamine receptor gene expression, and cellular responses to this amine are all affected by QMP. We identify homovanillyl alcohol as a key contributor to these effects and provide evidence linking QMP-induced changes in the brain to changes at a behavioral level. This study offers exciting insights into the mechanisms through which QMP operates and a deeper understanding of the queen's ability to regulate the behavior of her offspring.     

Age and natural metabolically-intensive behavior affect oxidative stress and antioxidant mechanisms

Flying honey bees have among the highest mass-specific metabolic rates ever measured, suggesting that their flight muscles may experience high levels of oxidative stress during normal daily activities. We measured parameters of oxidative stress and antioxidant capacity in highly metabolic flight muscle and less active head tissue in cohorts of age-matched nurse bees, which rarely fly, and foragers, which fly several hours per a day. Naturally occurring foraging flight elicited an increase in flight muscle Hsp70 content in both young and old foragers; however catalase and total antioxidant capacity increased only in young flight muscle. Surprisingly, young nurse bees also showed a modest daily increase in Hsp70, catalase levels and antioxidant capacity, and these effects were likely due to collecting the young nurses soon after orientation flights. There were no differences in flight muscle carbonyl content over the course of daily activity and few differences in Hsp70, catalase, total antioxidant capacity and protein carbonyl levels in head tissue regardless of age or activity. In summary, honey bee flight likely produces high levels of reactive oxygen species in flight muscle that, when coupled with age-related decreases in antioxidant activity may be responsible for behavioral senescence and reduced longevity.     

衰老心肌自噬减退的新机制-转录因子EB的关键作用

目的 探讨转录因子EB（TFEB）在衰老心肌自噬减退中的作用。方法 采用老年（22月龄）雄性C57BL/6小鼠为实验对象,以成年（4月龄）雄性C57BL/6小鼠为对照,分析心肌自噬水平、心肌TFEB表达水平。结果 与成年心肌相比,衰老心肌自噬水平显著降低（P<0.05）。衰老心肌中自噬体标志物Atg5、LC3和Beclin-1,溶酶体标志物LAMP1在蛋白和mRNA水平上均出现降低。与成年心肌相比,衰老心肌TFEB蛋白水平显著降低（P<0.05）,衰老心肌细胞核内的TFEB水平下降更为显著（P<0.05）,提示衰老心肌TFEB转录能力减退。给予小剂量雷帕霉素处理,可提高衰老心肌细胞核内TFEB水平,并且改善LC3及LAMP1的mRNA和蛋白水平,提高衰老心肌自噬水平。结论 本研究发现衰老导致的心肌TFEB水平降低严重影响心肌自噬能力,提示TFEB是心肌自噬增龄性减退机制中的关键调节因子。     

Reproductive protein protects functionally sterile honey bee workers from oxidative stress

Research on aging shows that regulatory pathways of fertility and senescence are closely interlinked. However, evolutionary theories on social species propose that lifelong care for offspring can shape the course of senescence beyond the restricted context of reproductive capability. These observations suggest that control circuits of aging are remodeled in social organisms with continuing care for offspring. Here, we studied a circuit of aging in the honey bee (Apis mellifera). The bee is characterized by the presence of a long-lived reproductive queen caste and a shorter-lived caste of female workers that are life-long alloparental care givers. We focus on the role of the conserved yolk precursor gene vitellogenin that, in Caenorhabditis elegans, shortens lifespan as a downstream element of the insulin/insulin-like growth factor signaling cascade. Vitellogenin protein is synthesized at high levels in honey bee queens and is abundant in long-lived workers. We establish that vitellogenin gene activity protects worker bees from oxidative stress. Our finding suggests that one mechanistic explanation for patterns of longevity in bees is that a reproductive regulatory pathway has been remodeled to extend life. This perspective is of considerable relevance to research on longevity regulation that builds largely on inference from solitary model species.     

Lack of hypergravity-associated longevity extension in Drosophila melanogaster flies overexpressing hsp70

Previous studies have shown that Drosophila melanogaster males live longer if exposed to hypergravity at young age. Similarly, thermotolerance and climbing activity in old age are enhanced and it has been hypothetized that the 70 kDa inducible heat-shock protein (hsp70) may play a role in some of these effects. In this article, longevity, thermotolerance and climbing activity were studied in transgenic flies (OE+strain) over expressing hsp70 and in control flies harboring the transfection vector at the same chromosomal location but no extra-copies of the hsp70 gene (OE−strain). No positive effect from exposure to hypergravity at a young age was observed for longevity and climbing activity. Thermotolerance was increased by hypergravity but to the same extent in OE+ and OE− strains. Since no positive effect from hypergravity exposure or similar effects were observed in the two strains, it may be concluded that over expression ofhsp70 does not provide an advantage to flies subjected to a mild stress and that it may not be of any help during aging. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced protein repair and recycling are not correlated with longevity in 15 vertebrate endotherm species

Previous studies have shown that longevity is associated with enhanced cellular stress resistance. This observation supports the disposable soma theory of aging, which suggests that the investment made in cellular maintenance will be proportional to selective pressures to extend lifespan. Maintenance of protein homeostasis is a critical component of cellular maintenance and stress resistance. To test the hypothesis that enhanced protein repair and recycling activities underlie longevity, we measured the activities of the 20S/26S proteasome and two protein repair enzymes in liver, heart and brain tissues of 15 different mammalian and avian species with maximum lifespans (MLSP) ranging from 3 to 30 years. The data set included Snell dwarf mice, in which lifespan is increased by ∼50% compared to their normal littermates. None of these activities in any of the three tissues correlated positively with MLSP. In liver, 20S/26S proteasome and thioredoxin reductase (TrxR) activities correlated negatively with body mass. In brain tissue, TrxR was also negatively correlated with body mass. Glutaredoxin (Grx) activity in brain was negatively correlated with MLSP and this correlation remained after residual analysis to remove the effects of body mass, but was lost when the data were analysed using Felsenstein’s independent contrasts. Snell dwarf mice had marginally lower 20S proteasome, TrxR and Grx activities than normal controls in brain, but not heart tissue. Thus, increased longevity is not associated with increased protein repair or proteasomal degradation capacities in vertebrate endotherms.     

Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae

Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5-/- cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.     

Selective downregulation of ubiquitin conjugation cascade mRNA occurs in the senescent rat soleus muscle

Aging-related alterations of the ubiquitin proteasome pathway (UPP) have been reported in locomotor skeletal muscle. Specifically, declines in proteasome activity have been observed in the soleus of senescent animals compared to the soleus of young controls. However, the influence of aging on the mRNA levels of key components within the ubiquitin conjugation cascade (UCC) remains unknown. We hypothesized that aged soleus muscle would exhibit downregulated expression of select UCC mRNA and decreased levels of ubiquitin-protein conjugates. To test this postulate, we harvested soleus muscles from 6 and 24-26 month old Fisher 344 rats. Aging resulted in a decline in mRNA expression of two key UCC components in soleus muscle; ubiquitin conjugating enzyme E2(14k) (E2(14k)) and muscle ring finger-1 (MuRF1). Surprisingly, no age-related differences existed in the total content of endogenous ubiquitin-protein conjugates in the soleus muscle. Nonetheless, a selective decrease in the level of ubiquitin-protein conjugates ( approximately 30kDa) was detected in the soleus of senescent animals. These results indicate that the soleus muscle displays a differential mRNA response of select UCC components to aging. Furthermore, the decline in E2(14k) and MuRF1 mRNA levels may contribute to altered substrate degradation by the UCC in the soleus muscle of senescent rats.