Selected contribution: Bone adaptation with aging and long-term caloric restriction in Fischer 344 x Brown-Norway F1-hybrid rats.

Rodents are commonly used as models for human aging because of their relatively short life span, the ease of obtaining age-specific tissue samples, and lower cost. However, age-associated disease may confound inbred animal studies. For example, numerous physiologically significant lesions, such as chronic nephropathy, are more common in aged Fischer 344 (F344) rats than in other strains (Bronson RT, Genetic Effects of Aging, 1990). Conversely, F344 x Brown-Norway F1-hybrid (F344BN) rats, developed by the National Institute on Aging for aging research, live considerably longer and have fewer pathologies at any given age vs. inbred strains (Lipman RD, Chrisp CE, Hazzard DG, and Bronson RT, J Gerontol A Biol Sci Med Sci 51: 54-59, 1996). To our knowledge, there are no data regarding the effect of age on bone geometry and mechanics in this strain of rat. Furthermore, caloric restriction (CR) extends the mean and maximal life span of animals and significantly reduces age-associated disease but may have adverse consequences for bone growth and mechanics. Thus we investigated the effects of age and CR on bone geometry and mechanics in the axial and appendicular skeleton of F344 Brown-Norway rats. Ad libitum fed rats were assessed at 8 mo (young adult; n = 6), 28 mo (late middle age; n = 5), and 36 mo (senescence; n = 6). CR rats were assessed at 28 mo (n = 6). Tibiae and the sixth lumbar vertebrae (L6) were dissected, scanned (micro-computed tomography) to determine geometry, and tested mechanically. From 8 to 36 mo, there were no significant changes in L6 geometry, and only the cross-sectional moment of inertia changed (increased) with the tibia. CR-induced body mass reductions accounted for changes in L6 load at proportional limit, maximal load, and stiffness (structural properties), but altered tibial structural properties were independent of body mass. In tibiae, geometric changes dominated alterations in structural properties. Those data demonstrated that, whereas aging in ad libitum-fed animals induced minor changes in bone mechanics, axial and appendicular bones were adversely influenced by CR in late-middle-aged animals in different manners.     

DEPTOR Cell-Autonomously Promotes Adipogenesis, and Its Expression Is Associated with Obesity

DEP domain-containing mTOR-interacting protein (DEPTOR) inhibits the mechanistic target of rapamycin (mTOR), but its in?vivo functions are unknown. Previous work indicates that Deptor is part of the?Fob3a quantitative trait locus (QTL) linked to obesity/leanness in mice, with Deptor expression being elevated in white adipose tissue (WAT) of obese animals. This relation is unexpected, considering the?positive role of mTOR in adipogenesis. Here, we?dissected the Fob3a QTL and show that Deptor is the highest-priority candidate promoting WAT expansion in this model. Consistently, transgenic mice overexpressing DEPTOR accumulate more WAT. Furthermore, in humans, DEPTOR expression in WAT correlates with the degree of obesity. We show that DEPTOR is induced by glucocorticoids during adipogenesis and that its overexpression promotes, while its suppression blocks, adipogenesis. DEPTOR activates the proadipogenic Akt/PKB-PPAR-γ axis by dampening mTORC1-mediated feedback inhibition of insulin signaling. These results establish DEPTOR as a new regulator of adipogenesis.     

Age-Related Modulation of the Effects of Obesity on Gene Expression Profiles of Mouse Bone Marrow and Epididymal Adipocytes

This study aimed to characterize and compare the effects of obesity on gene expression profiles in two distinct adipose depots, epididymal and bone marrow, at two different ages in mice. Alterations in gene expression were analyzed in adipocytes isolated from diet-induced obese (DIO) C57BL/6J male mice at 6 and 14 months of age and from leptin deficient mice (ob/ob) at 6 months of age using microarrays. DIO affected gene expression in both depots at 6 and 14 months, but more genes were altered in epididymal than bone marrow adipocytes at each age and younger mice displayed more changes than older animals. In epididymal adipocytes a total of 2789 (9.6%) genes were differentially expressed at 6-months with DIO, whereas 952 (3.3%) were affected at 14-months. In bone marrow adipocytes, 347 (1.2%) genes were differentially expressed at 6-months with DIO, whereas only 189 (0.66%) were changed at 14-months. 133 genes were altered by DIO in both fat depots at 6-months, and 37 genes at 14-months. Only four genes were altered in both depots at both ages with DIO. Bone marrow adipocytes are less responsive to DIO than epididymal adipocytes and the response of both depots to DIO declines with age. This loss of responsiveness with age is likely due to age-associated changes in expression of genes related to adipogenesis, inflammation and mitochondrial function that are similar to and obscure the changes commonly associated with DIO. Patterns of gene expression were generally similar in epididymal adipocytes from ob/ob and DIO mice; however, several genes were differentially expressed in bone marrow adipocytes from ob/ob and DIO mice, perhaps reflecting the importance of leptin signaling for bone metabolism. In conclusion, obesity affects age-associated alterations in gene expression in both epididymal and bone marrow adipocytes regardless of diet or genetic background.     

Aging differentially alters the expression of angiogenic genes in a tissue-dependent manner

Organ functions are altered and impaired during aging, thereby resulting in increased morbidity of age-related diseases such as Alzheimer’s disease, diabetes, and heart failure in the elderly. Angiogenesis plays a crucial role in the maintenance of tissue homeostasis, and aging is known to reduce the angiogenic capacity in many tissues. Here, we report the differential effects of aging on the expression of angiogenic factors in different tissues, representing a potentially causes for age-related metabolic disorders. PCR-array analysis revealed that many of angiogenic genes were down-regulated in the white adipose tissue (WAT) of aged mice, whereas they were largely up-regulated in the skeletal muscle (SM) of aged mice compared to that in young mice. Consistently, blood vessel density was substantially reduced and hypoxia was exacerbated in WAT of aged mice compared to that in young mice. In contrast, blood vessel density in SM of aged mice was well preserved and was not different from that in young mice. Moreover, we identified that endoplasmic reticulum (ER) stress was strongly induced in both WAT and SM during aging in vivo. We also found that ER stress significantly reduced the expression of angiogenic genes in 3T3-L1 adipocytes, whereas it increased their expression in C2C12 myotubes in vitro. These results collectively indicate that aging differentially affects the expression of angiogenic genes in different tissues, and that aging-associated down-regulation of angiogenic genes in WAT, at least in part through ER stress, is potentially involved in the age-related adipose tissue dysfunction.     

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.     

Development of progressive aortic vasculopathy in a rat model of aging

Recent studies have established that age is the major risk factor for vascular disease. Numerous aberrant changes occur in vascular structure and function during aging, and animal models are the primary means to determine the underlying mechanisms of age-mediated vascular pathology. The Fischer 344/Brown Norway F1 hybrid (F344xBN) rat thoracic aorta has been shown to display age-related pathology similar to what occurs in humans. This study utilized the F344xBN rat aorta and both morphometric and global gene expression analyses to identify appropriate time points to study vascular aging and to identify molecules associated with the development and progression of vascular pathology. In contrast to some previous studies that indicated age-related abrupt changes, a progressive increase in intimal and medial thickness, as well as smooth muscle cell-containing intimal protrusions, was observed in thoracic aorta. This structural vascular pathology was associated with a progressive, but nonlinear, increase in global differential gene expression. Gene products with altered mRNA and protein expression included inflammation-related molecules: specifically, the adhesion molecules ICAM-1 and VCAM-1 and the bone morphogenic proteins osteopontin and bone sialoprotein-1. Intimal-associated macrophages were found to increase significantly in number with age. Both systemic and tissue markers of oxidant stress, serum 8-isoprostane and 3-nitrotyrosine, respectively, were also found to increase during aging. The results demonstrate that major structural abnormalities and altered gene expression develop after 6 mo and that the progressive pathological development is associated with increased inflammation and oxidant stress.     

Age-associated differences in cardiovascular inflammatory gene induction during endotoxic stress

Upon physiological stress, families of stress response genes are activated as natural defense mechanisms. Here, we show that induction of specific inflammatory genes is significantly dysregulated and altered in the heart of aged (24--26-month-old) versus young (4-month-old) mice experimentally challenged with a bacterial endotoxin, lipopolysaccharide (LPS, 1.5 mg/kg of body mass). Whereas the LPS-mediated induction of cardiac mRNA for tumor necrosis factor alpha or inducible nitric-oxide synthase showed no age-associated differences, the induction of interleukin-1 beta (IL-1 beta) and intracellular adhesion molecule-1 was modestly extended with aging, and the induction of IL-6 was significantly prolonged with aging. This age-associated phenomenon occurred gradually from 4 to 17 months of age and became more evident after 23 months of age. The age-associated augmentation of the cardiac IL-6 induction was also dramatic at the protein level. Immunohistochemically, the LPS-induced cardiac IL-6 was localized mainly in the microvascular walls. Aged but not young mice showed a high mortality rate during these experiments. These results demonstrate that endotoxin-mediated induction of specific inflammatory genes in cardiovascular tissues is altered with aging, which may be causally related to the increased susceptibility of aged animals to endotoxic stress.     

Altered expression of C/EBP family members results in decreased adipogenesis with aging

Fat mass, adipocyte size and metabolic responsiveness, and preadipocyte differentiation decrease between middle and old age. We show that expression of CCAAT/enhancer binding protein (C/EBP)-alpha, a key regulator of adipogenesis and fat cell function, declined substantially with aging in differentiating preadipocytes cultured under identical conditions from rats of various ages. Overexpression of C/EBP alpha in preadipocytes cultured from old rats restored capacity to differentiate into fat cells, indicating that downstream differentiation-dependent genes maintain responsiveness to regulators of adipogenesis. C/EBP alpha-expression also decreased with age in fat tissue from three different depots and in isolated fat cells. The overall level of C/EBP beta, which modulates C/EBP alpha-expression, did not change with age, but the truncated, dominant-negative C/EBP beta-liver inhibitory protein (LIP) isoform increased in cultured preadipocytes and isolated fat cells. Overexpression of C/EBP beta-LIP in preadipocytes from young rats impaired adipogenesis. C/EBP delta, which acts with full-length C/EBP beta to enhance adipogenesis, decreased with age. Thus processes intrinsic to adipose cells involving changes in C/EBP family members contribute to impaired adipogenesis and altered fat tissue function with aging. These effects are potentially reversible.     

Hippocampal Gene Expression Meta-Analysis Identifies Aging and Age-Associated Spatial Learning Impairment (ASLI) Genes and Pathways

A number of gene expression microarray studies have been carried out in the past, which studied aging and age-associated spatial learning impairment (ASLI) in the hippocampus in animal models, with varying results. Data from such studies were never integrated to identify the most significant ASLI genes and to understand their effect. In this study we integrated these data involving rats using meta-analysis. Our results show that proper removal of batch effects from microarray data generated from different laboratories is necessary before integrating them for meta-analysis. Our meta-analysis has identified a number of significant differentially expressed genes across age or across ASLI. These genes affect many key functions in the aged compared to the young rats, which include viability of neurons, cell-to-cell signalling and interaction, migration of cells, neuronal growth, and synaptic plasticity. These functional changes due to the altered gene expression may manifest into various neurodegenerative diseases and disorders, some of which leading into syndromic memory impairments. While other aging related molecular changes can result into altered synaptic plasticity simply causing normal aging related non-syndromic learning or spatial learning impairments such as ASLI.     

Common mechanisms for calorie restriction and adenylyl cyclase type 5 knockout models of longevity

Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared with WT mice. CR applied to AC5 KO results in rapid decrease in body weight, metabolic deterioration, and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome‐wide for brain, heart, skeletal muscle, and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene ontology analysis identified many significantly regulated, tissue‐specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD, and olfactory receptors in both young‐ and intermediate‐age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR mice, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance.     

Chronic mTOR inhibition in mice with rapamycin alters T,B, myeloid,and innate lymphoid cells and gut flora and prolongs life of immune‐deficient mice

The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age‐related debilities including increasing antigen‐specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long‐term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T‐ and B‐lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer‐ and infection‐prone mice supporting disease mitigation as a mechanism for mTOR suppression‐mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension.     

Moderate caloric restriction in lactating rats programs their offspring for a better response to HF diet feeding in a sex-dependent manner

We aimed to assess the lasting effects of moderate caloric restriction in lactating rats on the expression of key genes involved in energy balance of their adult offspring (CR) and their adaptations under high-fat (HF) diet. Dams were fed with either ad libitum normal-fat (NF) diet or a 30% caloric restricted diet throughout lactation. After weaning, the offspring were fed with NF diet until the age of 15 weeks and then with an NF or a HF diet until the age of 28 weeks, when they were sacrificed. Body weight and food intake were followed. Blood parameters and the expression of selected genes in hypothalamus and white adipose tissue (WAT) were analysed. CR ate fewer calories and showed lower body weight gain under HF diet than their controls. CR males were also resistant to the increase of insulin and leptin occurring in their controls under HF diet, and HF diet exposed CR females showed lower circulating fasting triglyceride levels than controls. In the hypothalamus, CR males had higher ObRb mRNA levels than controls, and CR females displayed greater InsR mRNA levels than controls and decreased neuropeptide Y mRNA levels when exposed to HF diet. CR males maintained WAT capacity of fat uptake and storage and of fatty-acid oxidation under HF diet, whereas these capacities were impaired in controls; female CR showed higher WAT ObRb mRNA levels than controls. These results suggest that 30% caloric restriction in lactating dams ameliorates diet-induced obesity in their offspring by enhancing their sensitivity to insulin and leptin signaling, but in a gender-dependent manner.