Gut neuroendocrine cells: relationship to the proliferative activity and apoptosis of mucous epitheliocytes in aging.

OBJECTIVES: Diffuse neuroendocrine system (DNES) cells regulate homeostasis via neurocrine, endocrine and paracrine mechanisms. Extensive effects of peptide hormones and biogenic amines necessitate studying of DNES cell biology in aging. In this connection, the functional morphology of gut neuroendocrine cells (NEC), proliferative activity and apoptosis of mucous epithelial cells in aging have been studied. MATERIAL AND METHODS: The study was performed on BALB/c-nu mice of 4, 21 and 34 months of age. NEC, proliferative activity and apoptosis of mucous epitheliocytes in stomach and duodenum have been studied by histochemical, immunohistochemical and morphometrical methods. RESULTS: The total number of NEC shows an increasing trend with advancing age. However, the different types of NEC elicit differential patterns. The total number of epithelial cell nuclei does not show any statistically significant difference during aging. The proliferative activity of mucous epitheliocytes also shows no difference among the three animal groups studied. On the contrary, the apoptotic index increases with advancing age. CONCLUSIONS: The results demonstrate that various gut NEC show differential behavior with age and their time-courses are dependent on the site of location (stomach or duodenum). The picture seems quite complex to allow a comprehensive interpretation, nonetheless it gives us some useful indications for further investigation. In fact, since the gut does not show evident gross age-related physiological changes, modifications with age in specific biological parameters can suggest the key mechanisms of compensative regulatory processes possibly acting during aging.

Mathematical modeling of the aging processes and the mechanisms of mortality: paramount role of heterogeneity.

Main problems of modeling the link between aging processes and mechanisms of mortality are addressed. Various applications of Gompertz's law, which allowed to formulate some fruitful hypotheses on the field, are reviewed. Some pitfalls occurring in its applications are also discussed using a model built on purpose to overcome these difficulties. The role played by heterogeneity emerges as the common cause of some relevant failure in using Gompertz's law and the necessary key ingredient of any model aimed to interpret the link between aging and mortality correctly. Though a number of problems are related to inter-individual variability, the search for their solution can lead to an intriguing approach to the study of aging and mortality. Living beings can be considered as complex systems and their age-related changes can be described at the light of complex system theory.

Use of mathematical models of survivorship in the study of biomarkers of aging: the role of heterogeneity.

An ever increasing number of people have been engaging in aging research using various interventions aimed to modify aging processes, and/or life span, of experimental animals. Since this type of studies needs outcome parameters for assessing the efficacy of such interventions, research on biomarkers of aging (ABs) has received new stimuli. In the present paper, the problem of the occurrence of a vicious circle any time we study ABs and determinants of aging is addressed. In fact, while ABs would represent the standard reference to be used in the study of the main causes of processes of aging, these very determinants should already be known in order to get reliable ABs. A feasible way to overcome this impasse is proposed, using mathematical models of survivorship or mortality based on biological hypotheses and accounting for inter-individual heterogeneity, a necessary ingredient for a correct interpretation of survival results. Specific kinetics of experimental parameters that are candidates as ABs can be compared to the kinetics hypothesized for general biological functions entering the model. We have built a model of this type that can also be used to perform a reliable overall gross estimate of the rate of aging, R(a), in the population, a parameter useful when judging the success of interventions aimed to act on determinants of aging. The perspective that theory of complex systems can be of help in the search for ABs is also discussed.

Long-live euthymic BALB/c-nu mice. I. Survival study suggests body weight as a life span predictor.

This paper is the first of a series aimed to show the main physiological and pathological characteristics of male euthymic BALB/c-nu mice, a long-live strain of BALB/c mice bred in our own Institute. In particular, the first two paired papers are respectively devoted to general survival information and disease characteristics, also taking into account very old animals that are of high interest for studies on successful aging. In this paper we report the analysis of survival kinetics, the time course of body weight and the correlation between body weight and time-at-death. The longitudinal study has been performed on 88 male mice, checking individually their body weight and date of death and analyzing survival data by a model built by our own. Survival analysis shows quite higher longevity (median age: about 29 months) in this population when compared with other BALB/c strains. The most relevant finding on body weight is its correlation with longevity until the age of 22 months: thinner subjects live longer and lose weight at a lower rate than their heavier mates. Results have formed the basis on which to plan the cross-sectional experiment to study pathologies and biological parameters at different ages, including a group of mice at very advanced ages (34 months).

Long-live euthymic BALB/c-nu mice. II: spontaneous tumors and other pathologies.

This paper is the second of a series aimed to show the main physiological and pathological characteristics of male euthymic BALB/c-nu mice, a long-live strain of BALB/c mice bred in our own Institute. The previous paired paper Piantanelli (Mech. Ageing Dev. (2001)) has been devoted to a survival study up to advanced ages highly interesting for studies on successful aging. In the present paper we report first data of a cross-sectional study on 4,15,22,28 and 34 months-old mice, dealing with tumors and other relevant pathologies. Results have shown that tumors or other pathologies can hardly be detected up to the age of 22 months. At 34 months of age about 40% of mice revealed a variety of neoplasia and other diseases are clearly detectable. These results suggest that a significant increase in longevity could be a factor increasing the risk of tumor development; thus, caution has to be paid in studies on mice utilized for long term carcinogenicity assay, where animals are sacrificed at the age of 18 months, according to the International Program. Finally, animals of the same chronological age have been subdivided in clusters according to their presumptive longevity, estimated taking advantage of the relationship between body weight and age-at-death found in the paired longitudinal study. This subdivision will be helpful in interpreting inter-individual variability of the biological parameters checked in these animals.

Transcriptional regulation of the androgen signaling pathway by the Wilms' tumor suppressor gene WT1.

The androgen-signaling pathway plays a critical role in prostate cancer development and progression. We have recently demonstrated that the Wilms' tumor suppressor gene product, WT1, binds to multiple sites in the androgen receptor (AR) promoter and transcriptionally represses the AR gene promoter in vitro. We asked whether WT1 repression of the endogenous AR gene interferes in the androgen signal transduction cascade and modifies AR target gene expression. We analyzed the effect of WT1 (-/-) overexpression on an AR target gene reporter construct that contains the luciferase gene, the ElB TATA box, and two copies of the androgen-response element (ARE), the dimeric AR binding site. Luciferase activity was determined in 293 kidney and TM4 Sertoli cells, two nontumorigenic cell lines that express both AR and WT1. Cells were cotransfected by lipofectamine in the presence or absence of the synthetic androgen R1881. Results showed that overexpression of WT1 downregulates ARE-reporter gene transcription in both cell lines tested. The inhibitory effect of WT1 on the AR target gene construct was dose-dependent and androgen-independent in 293 cells, whereas in TM4 cells it was androgen-dependent. Additionally, a zinc-finger mutant WT1 (-/-) expression construct, R394W, failed to decrease luciferase activity, suggesting that WT1 downregulates the ARE-reporter gene construct activity by directly repressing the endogenous AR gene promoter. Furthermore, we analyzed the expression of WT1 and AR mRNA in several prostate cancer cell lines in order to understand the role WT1 may play in prostate cancer development and progression. Gel analysis of cDNA amplified by RT-PCR of AR and WT1 RNA from prostate cancer and non-prostatic cell lines showed that LNCaP and MDAPCa2b, two metastatic prostate cancer cell lines which are androgen-sensitive, expressed AR but not WT1. Du145 and PC3, two cell lines from advanced metastatic prostate cancer, which are characterized as androgen-independent and -insensitive, did not express AR but expressed a high level of WT1. Two non-prostatic cell lines, T47D and 293, weakly co-expressed AR and WT1. This inverse relationship between AR and WT1 expression in prostate cancer cell lines, together with WT1 repression of the AR promoter, suggest a role for WT1 in the androgen signaling pathway and in prostate cancer development and progression.

Excitatory amino acid receptors in the prefrontal cortex of aging mice.

The zones of the prefrontal cortex of Balb/c mice were tested for age-related changes of the ionotropic excitatory amino acid receptors density, together with zones of the dorsal cortex. Kainate, N-methyl-D-aspartate, and amino-3-hydroxy-5-methyloxazole-4-propionate sites were measured by slice receptor binding techniques in cortical zones from animals at the age of 6, 12, 18, and 24 months. An increase of the N-methyl-D-aspartate sites was detected in the medial prefrontal zone of mid-aged animals and was followed by a decrease at old age; a decrease of the N-methyl-D-aspartate and kainate sites was found for the medial dorsal (cingulate) cortex at old age. The age-related changes of receptor densities in the different cortical areas seem unrelated in origin. The sites decrease in the cingulate cortex could affect the transfer of the prefrontal cortex activity toward limbic structures.

Neurochemical parameters of the main neurotransmission systems in aging mice.

The present work was designed to study the effect of aging on some parameters of the glutamatergic, aminergic and cholinergic neurotransmission, in the main brain areas of mice of the long-surviving BALB/c-nu strain. We have assayed: (1) the density of three ionotropic receptors for excitatory aminoacids (EAA) which selectively bind kainic acid (KA), N-methyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA); (2) the content of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) and the levels of the DA metabolite dihydrophenylacetic acid (DOPAC) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA); (3) the level of the choline acetyltransferase (ChAT), the enzyme catalyzing the synthesis of acetylcholine. The parameters were measured in animals at the age of 6, 12, 18 and 24 months; the brain zones under test were the frontal cortex (FC), the corpus striatum (STR), the hippocampus (HIP), the medio-dorsal cortex (DC) and the cerebellum (CER). Significant age-related variations for the density of KA-type and NMDA-type receptors were found in STR and a decrease of the NMDA parameter was found in DC. Neither the monoamine and metabolite contents nor the ChAT levels showed any significant variation in all the tested areas. These findings suggest that an unbalance among different neurotransmission activities could take place with normal aging in rodents: it could be involved in the onset of the motor deficit which occurs in the elderly of these and other mammals.

Insulin receptors in the brain cortex of aging mice.

The aim of the present study was to analyze whether aging also affects central insulin receptors in brain cortex as it does in whole brain of BALB/c-nu mice. Results showed statistically significant decrease of number and increase of affinity of insulin high affinity binding sites in old animals. As a consequence, central insulin actions, among which neuromodulation of monoaminergic system, can result altered during aging.

Insulin receptors in mouse brain: Reversibility of age-related impairments by a thymic extract.

Recently, we have shown that insulin receptors (InsRs) in the brain undergo impairments with aging. Interestingly, age-related alterations of brain InsRs, are not irreparable as thymus grafts are able to recover them. With the present study we verified the possibility that an aqueous extract from calf thymus (TME) can mimic the restoring action of age-related impairments induced by thymus graft. InsR characteristics were assayed in a group of 25 months old BALB/c-nu mice treated with TME: 2µg/g body weight every third day, for total five subcutaneous injections. The last dose was injected the day before animals were killed. Other two groups of young (4 months) and old (25 months) mice received saline solution with the same schedule. A two-sites model analysis of receptor data confirms the age-dependent decrease of InsR number and kd previously observed in the high affinity population. Furthermore, a statistically significant recovery of number impairment is shown in TME-treated animals. On the contrary, the characteristics of the low affinity receptor subset show no statistically significant differences among the three animal models studied. TME induced recovery of the age-related changes found in brain InsRs, together with previously observed regulatory action of the same thymic extract on the adrenergic system, suggest that thymic gland does not necessarily have to mutually interact with other controlling systems for maintaining or recoving homeostasis of the complex neuroendocrine network during development and aging.

Modulation of mouse brain cortex adrenoceptor in old mice by supplementation of zinc and thymomodulin.

BACKGROUND: Previous experiments have shown that the age-related decrease of mouse brain cortex adrenoceptor density can be recovered by grafting a neonatal thymus into old recipients. The question arises whether similar results can also be obtained in the presence of a single thymic factor such as thymomodulin (TMD). It is worth noting that the activity of some thymic factors is strictly zinc (Zn) dependent and that their age-related decreased production is recovered in old mice supplemented with Zn. OBJECTIVE: The above-mentioned evidences prompted us to verify whether Zn and TMD, either alone or combined, are able to induce some corrective effects on age-dependent alterations of adrenoceptor characteristics of the mouse brain cortex. METHODS: Thus, we performed experiments on four groups of Balb/c mice treated with saline, Zn, TMD, or both Zn and TMD. Treatments started when animals were 18 months old and ran for 6 months. The alpha(1)- and beta-Adrenoceptor characteristics were assayed by steady state binding analysis using labelled prazosine and iodocyanopindolol, respectively. Data were analyzed using one-way analysis of variance, followed, when appropriate, by multiple-comparison analysis. RESULTS: Results show an increase of beta-adrenoceptor density and a decrease of alpha(1)-adrenoceptor density in both Zn- and Zn+TMD-treated animals when compared to saline-treated controls, while receptor affinities did not change significantly. CONCLUSIONS: The lack of action of TMD suggests that this type of treatment cannot mimic the effects of grafting the whole gland; it cannot be excluded that different time-dose schedule could be more effective. Zn treatment, on the other hand, does modulate adrenoceptors; however, it shows a corrective action on the age-related decreased density of beta-adrenoceptors, but further decreases that of alpha(1)-adrenoceptors. This differential action could be due to their differential physiological role.

Reduced DNA synthesis in primary cultures of hepatocytes from old mice is restored by thymus grafts.

We previously observed in vivo that a neonatal thymus grafted into old mice can correct age-related changes such as occurrence of hepatocyte tetraploid nuclei and impaired isoproterenol-induced DNA synthesis in submandibular glands. The aim of the present paper was to study the influence of age and thymus on basal and beta-adrenergic-stimulated DNA synthesis using primary cultures of mouse hepatocytes. In the absence of any adrenergic agents, cells from young mice show peak DNA synthesis between 36 and 48 h; old mice show a similar time course, but the peak is significantly reduced statistically. The main result is represented by the behavior of hepatocytes from old thymus-grafted mice, which recover the levels of [3H]-thymidine incorporation toward young-like values. Grafted animals also show a correction of total DNA content that is increased in old mice. The addition of isoproterenol does not modify the DNA synthetic pattern, whereas the antagonist propranolol causes a slight but statistically significant decrease.

Insulin receptors in mouse brain: age-related modifications are corrected by thymus graft.

Recently, we have shown that insulin receptors (InsRs) in the brain undergo impairment with aging, as happens for other receptors such as alpha- and beta-adrenoceptors. Age-related alterations of adrenoceptors, which are modulated by brain InsRs, are not definitive as they can be recovered by a thymus graft. In this study we verified the possibility that the thymus graft can also recover the age-dependent modifications of brain InsRs. InsR characteristics were assayed in a group of 27 months old Balb/c-nu mice grafted with a neonatal thymus, under renal capsule, one month before the animals were killed. Another two groups of young (3 months) and old (27 months) mice were used as controls. A two-sites model analysis of receptor data confirmed the age-dependent decrease of InsR density previously observed in the high affinity population. Furthermore, a statistically significant recovery of this impairment was shown in thymus grafted animals. The low affinity receptor subset also showed some differences among the three animal models; however, they were not statistically significant. Thymus graft induced recovery of the age-related changes found in brain InsRs, together with the similar one observed on the adrenergic system, calls for deeper studies of their interaction and the role they can play on aging processes.

Modelling survival kinetics for red blood cells.

In the attempt to improve the analysis of red blood cell survival kinetics we evaluated the ability of a new mathematical model of survivorship in fitting hemolysis curves. This model contains two parameters omega and S0 related to deterministic and stochastic components of mortality kinetics, respectively. In this paper, firstly, we show that the model can be usefully applied in the analysis of hemolysis kinetics of very different life span and shape. Then, we check the capability of fitting the model to experimental lysis curves derived from human erythrocytes incubated at different temperatures: our results demonstrate that there is good agreement between experimental and theoretical data.

Thyroid-thymus interaction on beta-adrenoceptor regulation during development and ageing.

Up-regulation of brain cortex beta-adrenoceptors (beta ARs) can be induced by very acute stimulation with a single injection of T4 or T3 in young Balb/c-nu mice. We have also shown that this very rapid receptor increase can also be demonstrated in ageing animals when stimulated with T3 but not T4 injection. The aim of the present paper was to verify the capability of the thymus to reverse these impairments which we often observed in other experiments on old mice and young athymic nudes. The up-regulation induced by T4 and T3 was studied in normal and athymic nude young, normal old, and normal old and athymic nude young mice grafted with neonatal thymus 1 month earlier. In addition, since brain cortex bears both beta AR subpopulations, the eventual differential behavior of beta 1AR and beta 2AR subtypes was also investigated. Animals were injected intraperitoneally with saline, or saline containing 0.5 microgram T3 or 6.4 micrograms T4 per g body weight and killed 15 or 60 min after injection. Results show that thymus can recover the modifications of basal levels as well as T3-induced up-regulation of beta ARs in nude and old mice. On the contrary, impaired response to T4 stimulation is corrected in nude but not in old mice. The peripheral conversion of T4 into T3 can explain their differential influence since a correct conversion only occurs in presence of an efficient beta-adrenergic function. Thus, a vicious circle may occur with a decreasing number of beta-adrenoceptors causing in old age altered T4 to T3 conversion, in turn responsible for altered beta-adrenergic responsiveness to T4.

Assay of total protein kinase activity in mouse brain cortex.

In previous studies we demonstrated that the adrenergic system is impaired in old animals and that the main alterations were observed at the level of receptor density and adenylate-cyclase activity. The decreased ability to produce cAMP could influence the activity of the cAMP dependent protein kinase (PKA), one of the enzymes responsible for the phosphorylation of protein substrates. Since protein phosphorylation is one of the most common and important mechanisms through which a cell regulates its activity, the characteristics of the phosphorus incorporation reaction were studied. Kinase activity was measured in homogenate of young mouse brain cortex prepared avoiding gross manipulations in order to maintain conditions as close to those present in the living animal as possible. Results show that phosphate incorporation is proportional to protein content and strictly dependent on ATP availability. Increasing the ATP concentration from 10 to 500 mumol/l, the length of incorporation phase increases, suggesting that the limiting point of the reaction is better represented by energy availability than by enzyme or protein substrate concentrations.

Alterations of brain insulin receptor characteristics in aging mice.

An interesting role of insulin and insulin receptors (InsRs) in the brain is neuromodulation of monoaminergic systems. Since our previous studies showed age-dependent alterations of alpha- and beta-adrenoceptors in mouse brain cortex, the intriguing role of brain InsRs per se and their involvement in adrenergic modulation prompted us to check their eventual changes with aging. Thus, brain InsR characteristics were studied in young (3 months) and old (27 months) Balb/c-nu mice by direct binding with (125)I-insulin. A two-sites model analysis of data shows a statistically significant age-related decrease of receptor density (39%) and k(d) (57%) in the high affinity population. The low affinity receptor subset also shows a decreasing trend of its characteristics; however, differences are not statistically significant and show a high degree of interindividual variability in both groups of mice.

Relationship between plasminogen activator inhibitor type-1 plasma levels and the lipoprotein(a) concentrations in non-insulin-dependent diabetes mellitus.

The first part of the paper deals with the relationship between two inhibiting factors of the complex enzyme cascade regulating fibrinolysis, namely plasminogen activator inhibitor type-1 (PAI-1) and lipoprotein(a) (Lp(a)). Blood concentrations of Lp(a), PAI-1 antigen (PAI-1 AG) and activity (PAI-1 AT), and the main parameters of lipo- and glyco-metabolic balance were studied in 80 type II diabetic patients. Roughly hyperbolic patterns have been found between PAI-1 and Lp(a). Negative statistically significant linear correlation can be elicited when Log PAI-1 AG and Log PAI-1 AT values are plotted versus Lp(a) values, the first one being particularly tight. These findings suggest a nearly on/off control of the two parameters, limiting the risk of hypofibrinolysis. The second part of the paper was aimed at verifying this hypothesis. A group of 30 diabetic patients were treated for 3 months with metformin, an antidiabetic biguanide compound which has been reported to reduce PAI-1 levels both in diabetic and in non-diabetic patients. Metformin significantly reduced PAI-1 AG and PAI-1 AT but did not influence plasma Lp(a) levels. A clear linear correlation between the basal Lp(a) values and the changes in PAI-1 AG levels was found. An even tighter correlation was elicited between the decrease in PAI-1, and PAI-1 pretreatment values.

Age-related regulation of mouse brain cortex adrenoceptors following camphor vapor exposition.

The paper deals with the ability of adrenergic receptors (AR) of mouse brain cortex to be differentially regulated in response to single or multiple expositions to camphor vapor. The regulation of alpha- and beta-adrenoceptors has been studied in young and old Balb/c-nu mice. Results confirm the decrease of total beta-adrenoceptor density previously observed in untreated mice with advancing age; in addition, receptor density decreases in both young and old mice after a single exposition to camphor vapor, followed by an adaptation after multiple stimuli. The beta1, subtype is mainly responsible for density decrease in young animals, while both beta1, and beta2 subtypes contribute to the decrease in old mice. On the contrary, beta2 subpopulation gives the major contribution to the adaptive recovery in both young and old mice. alpha-Adrenoceptors also show an age-related decrease in the control group; after a single exposition they show an increased density with the exception of alpha1-subset in the young group. Repeated expositions lead to a rather general adaptive response towards pre-stimuli conditions. The differential behaviour of receptor subtypes in response to camphor vapor exposition can be related to the differential alterations of receptor characteristics observed during aging and also suggests a possible mechanism through which these alterations may occur.