Fenofibrate mildly stimulates browning-associated expression in white adipose tissues of young but not old male rats.

The aim of this study was to examine the effects of the hypolipemic drug fenofibrate (FF) and aging on the expression of factors/enzymes involved in brown adipose tissue (BAT) function and browning of white adipose tissue epididymal (eWAT) and subcutaneous (sWAT) depots. Young-adult and old male Wistar rats were fed standard chow (control) or supplemented with 0.1% or 0.5% FF for 30 days. Tissue samples were analysed for gene expression and protein content, and stained with Oil Red O or hematoxylin and eosin. In BAT of young rats, 0.5% FF increased only Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 1 (CITED1) protein content and Fgf21 and Gpr109A mRNA expression. The expression of oxidative metabolism related genes (Pgc1α, Cpt1b, Mcad) decreased after 0.5% FF. In BAT of old rats, FF did not affect UCP1 and CITED1 content and had little effect on gene expression. Oil Red O staining of BAT revealed no changes in lipid droplet area upon treatment in either age group. In eWAT of young rats, 0.1FF elevated UCP1 protein content and Ucp1, Pgc-1α, and Mcad expression, whereas 0.5% FF increased PPARα content and Pgc-1α, Cpt1b, Mcad, and Gpr109A levels. In eWAT of old rats, only 0.1FF increased Pgc1α and Mcad expression. In both age groups median cell area of eWAT adipocytes was reduced after 0.5% FF. In sWAT Ucp1 gene expression was very low and UCP1 protein was undetectable. FF upregulated Ucp1, Cited1, Eva1, and Cpt1b expression in sWAT of young rats, with diminished effects in old rats. In both age groups 0.5% FF increased Fgf21 expression in sWAT. Median cell area of sWAT adipocytes decreased only in young rats treated with 0.5% FF. Our results reveal that fenofibrate differentially affects gene expression in BAT, with diminished effects in old compared to young rats. In WAT of young rats FF modestly stimulates the expression of factors/enzymes involved in lipid oxidative metabolism and browning. Aging reduces both these effects. Gpr109A may present a novel gene target upregulated by FF in BAT and eWAT.

Ultrastructural response of arcuate nucleus neurons to fasting in aged rats.

The arcuate nucleus of the hypothalamus (ARH) is involved in the control of energy homeostasis. Leptin - an adipocyte derived hormone - is known to act on the hypothalamic nuclei and thus to control body weight by food intake reduction. Oxidative stress is believed to be implicated in leptin signalling. However, its relevance for leptin-induced signal transduction within ARH remains unclear. The goal of the study was to investigate the effect of fasting on morphological alterations of the neuronal endoplasmic reticulum/Golgi network as well as on the expression of leptin receptors in the arcuate nucleus of aged rats. Male Wistar rats, aged 24 months, were fasted for 96 hours. The control animals were fed ad libitum. Membranous whorls in the ARH neurons were visualized using the electron microscopy technique. Leptin receptors in the membranes of ARH neurons were determined immunohistochemically (IHC), and soluble leptin receptors in the plasma as well as plasma isoprostanes were quantified immunochemically (ELISA). An intense formation of membranous whorls was observed, directly associated with the cisternae of the rough endoplasmic reticulum, as well as lamellar bodies. Interestingly, the whorls were often localized near a well-developed Golgi complex. Moreover, some Golgi complexes displayed an early stage of whorl formation. Groups of residual lipofuscin granules were found in the immediate proximity of the whorls. An increased immunoreactivity with neuronal leptin receptors suggests that hypersensitive neurons may still effectively respond to the fasting serum levels of leptin, mediating ultrastructural transformation of ARH neurons during short-term fasting. Having observed a significant accumulation of lipofuscin granules and a marked increase of total 8-isoprostane serum level in the fasting rats, we hypothesize that signal transduction within the neurons of ARH is dependent on oxidative stress phenomena.

Cell surface proteoglycan expression during maturation of human monocytes-derived dendritic cells and macrophages.

Cell surface proteoglycans play an important part in the functional and metabolic behaviour of leucocytes. We studied the expression of cell surface proteoglycans in human monocytes, in monocyte-derived immature and mature dendritic cells and in macrophages by metabolic labelling with [(35)S]-sulphate, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Immature dendritic cells had the highest metabolic activity for the synthesis of cell surface proteoglycans. The major part of these proteoglycans was in phosphatidylinositol-anchored form and was released after treatment with phospholipase C. A minor part was released by trypsin. Digestion with chondroitinase ABC and mild HNO(2) treatment showed that cell surface proteoglycans had a higher proportion of chondroitin sulphate, both in the phospholipase C and trypsin fractions, suggesting that at least some glypicans contained chondroitin sulphate chains. RT-PCR detected the transcripts of glypicans 1, 3, 4 and 5 and all syndecans. Immature dendritic cells expressed a most complex spectrum of glypicans and syndecans, glypican-1 and syndecan-1 being expressed preferentially by this type of cells. Mature dendritic cells expressed glypican-3, which was not present in other lineages. These results suggest that different mononuclear cells synthesize cell surface proteoglycans actively with characteristic expression of different syndecans and glypicans genes, depending on the degree of cell differentiation and/or maturation.

The effects of fasting and refeeding on adrenal cortex morphometry and serum concentrations of ACTH and corticosterone in young and old male rats.

The impairment of homeostatic mechanisms in ageing becomes often apparent upon physiological or pathological stimulation. We have previously shown that fasting and refeeding revealed the existence of age-related changes of carbohydrate and lipid metabolism. Because fuel metabolism is partially controlled by corticosteroids we decided to determine the effects of refeeding on adrenal gland morphometry, ACTH, and corticosterone serum levels in young (5 mo) and (20 mo) old male Wistar rats. Fasting for 48 h did not change serum ACTH and corticosterone in both age groups. ACTH level did not change after 24 h of refeeding in young and old rats. However, in old, but not young animals, refeeding resulted in the decrease of corticosterone serum concentration. The relative weight of adrenal gland (% of body weight) did not change significantly with age (p=0.05). Fasting for 48 h induced in old rats but not in young ones increase of relative adrenal weight, and the volume of the reticular zone. Refeeding reduced adrenal volume, fascicular zone and reticular zone. Refeeding for 24 h decreased the total volume of adrenal gland of old rats due to a decline of the volumes of fascicular and reticular zones. In young rats refeeding reduced the volume of reticular zone. It is concluded that refeeding revealed ageing-dependent decline in the secretion of corticosterone, the key hormone of prolonged stress response.

Thyroid C cell function during fasting and refeeding of young and old rats.

Age-related alterations in the structure and function of many organs often become apparent under stimulation of their function. Although the ageing process affects the regulation of mineral homeostasis, the function of thyroid C-cells that secrete calcitonin (CT) under the conditions of fasting and refeeding, a way of dietary manipulation that reveal the existence of age-related changes of follicular thyroid cells, has not been characterized. Therefore, we investigated the number of C-cells and serum CT concentration in young (4 mo) and old (26 mo) male rats fasted for 48 hours, and then refed for 4 or 24 hours. We found significantly higher number of C-cells in thyroids of old vs young rats both under basal conditions, and after fasting/refeeding. Correspondingly, serum calcitonin level was higher in fed or fasted old rats vs young ones. However, in young rats refeeding decreased, whereas in old animals increased serum concentrations of calcitonin. Thus, the control of serum calcium concentration, that was well preserved in old rats, occurs at the expense of increased serum CT level both under basal conditions, and after refeeding. These observations suggest that C-cell function is altered in ageing.

Natural killer activity and thyroid hormone levels in young and elderly persons.

BACKGROUND: On the basis that (1) multiple interactions exist between the hormonal and immune systems, and (2) aging is accompanied by changes in thyroid hormone metabolism and responsiveness, we postulate that thyroid hormones may be involved in the observed decrease in natural killer (NK) activity in a population of apparently healthy elderly subjects. The purpose of the study is to compare NK cytotoxic activity and serum concentrations of TSH and thyroid hormones in healthy old and young people, and to assess in vitro the effects of triiodothyronine (T(3)) on NK activity. MATERIALS AND METHODS: Sixteen of the 47 healthy old people (mean age 64 +/- 5.2) were classified as optimally healthy, and the remainder as 'almost healthy' (according to the criteria of the Senieur protocol) [Ligthart et al., Mech Ageing Dev 1984;28:47-55]; the mean age of the healthy young people was 23.3 +/- 2.3 years. NK cytotoxic activity of peripheral blood mononuclear cells was assessed using (51)Cr release from K562 target cells. The cutoff level for defining low and high NK responses was set at a value of 20%. Serum concentrations of TSH, total thyroxine (T(4)) and total triiodothyronine (T(3)) were measured by radioimmunoassay. RESULTS: NK activity in the 'optimally healthy' elderly was high (mean 41 +/- 12%, SE), whereas 'almost healthy' subjects showed low NK activity (mean 6 +/- 5%). Serum T(4) and TSH levels, but not T(3) concentrations were similar in both the young and old. We observed a significant correlation (r = 0.53, n = 21, p < 0.05) between the serum total T(3) level and the NK activity in the elderly individuals. Under in vitro conditions exogenous T(3) significantly increased NK activity in the elderly subjects who had serum T(3) values at the lower end of the reference range. However, no effect of T(3) on NK activity was observed in peripheral blood mononuclear cells obtained from either old or young individuals who had serum T(3) levels at the midpoint of the range. CONCLUSION: Decreased serum concentrations of total T(3) may contribute to low NK activity in the 'almost healthy' subgroup of the elderly.

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix.

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with 35S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher Mr than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.

The effect of fasting and refeeding on thyroid follicule structure and thyroid hormone levels in young and old rats.

In order to assess morphological and functional plasticity of the thyroid gland in aging the effects of fasting and refeeding on the thyroid morphology and thyroid hormone serum levels were compared with morphometry and RIA in young and old rats. Young (4 months) and old (24 months) male Wistar rats were fasted for 40 h and sampled, or fasted and fed thereafter for 4 or 24 h. In control (fed) old animals the thyroid follicules were larger, the follicular epithelium was smaller and colloid resorption was smaller than in young rats. 'Thyroid activation index' (epithelial volume/colloid volume ratio) was almost twice lower in the thyroids of control old rats. As the result of fasting, height, surface area and volume of epithelial follicular cells decreased in the thyroids of fasted young rats but not in old ones. On the contrary, in thyroids of fasted old rats the dimensions of epithelial cells did not change and thyroid colloid resorption was increased. After 24 h of refeeding, thyroid morphology in both young and old rats did not differ significantly as compared with control animals. Upon fasting, serum levels of thyroxine (T4) and triiodothyronine (T3) decreased by 28 and 38% in young and by 35 and 46% in old rats, respectively. However, T4 and T3 serum concentrations did not differ significantly between age groups in both fed and fasted states. During refeeding the increase in serum thyroxine concentration was delayed in old rats as compared with young ones. The results of morphological, morphometric and hormonal investigations indicate the existence of age-related changes in the structure and function of thyroid follicular cells.

Thyroid hormones homeostasis in rats refed after short-term and prolonged fasting.

Effects of starvation on thyroid hormone homeostasis were usually determined after 2 or more days of fasting, however, both in man and in rodents, natural feeding cycles comprise far shorter fasting periods. Therefore serum levels of T4, FT4, T3, FT3 and rT3 were measured in rats refed chow diet for 1, 4, 8 or 24 hours after 14 or 48 hours of starvation. Both short-term (14 h) and long-term fasting (48 h) decreased body weight and serum glucose level. Short-term fast decreased serum FT3 and did not change serum levels of T4, FT4, T3 and rT3. Total T3 and reverse T3 increased after one and 4 hours, free T3 after 4 hours and total T4 after 4 and 8 hours of refeeding. Percent of FT3 did not change after short-term fast, declined after 1 and 4 hours of refeeding, and normalised thereafter. Prolonged starvation (48 h) decreased serum T4, T3, FT3 and % FT3 with no changes in FT4 and rT3. After 24 hours of refeeding only FT3 and % FT3 returned to control levels while total T4 and total T3 were still diminished, and reverse T3 levels did not change. The results suggest that the length of preceding fasting period may strongly influence thyroid hormone homeostasis during fasted-to-fed transition.