Ghrelin-induced food intake and adiposity depend on central mTORC1/S6K1 signaling.

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) and its effectors the S6-kinases (S6K) in the hypothalamus is thought to be involved in nutrient sensing and control of food intake. Given the anatomical proximity of this pathway to circuits for the hormone ghrelin, we investigated the potential role of the mTORC1/S6K pathway in mediating the metabolic effects of ghrelin. We found that ghrelin promoted phosphorylation of S6K1 in the mouse hypothalamic cell line N-41 and in the rat hypothalamus after intracerebroventricular administration. Rapamycin, an inhibitor of mTORC1, suppressed ghrelin-induced phosphorylation of hypothalamic S6K1 and increased food intake and insulin in rats. Chronic peripheral administration of ghrelin induced a significant increase in body weight, fat mass and food efficiency in wild-type and S6K2-knockout but not in S6K1-knockout mice. We therefore propose that ghrelin-induced hyperphagia, adiposity and insulin secretion are controlled by a central nervous system involving the mTORC1/S6K1 pathway.

Effects of ghrelin on the structural complexity of exocrine pancreas tissue architecture.

Recent studies have shown that ghrelin increases pancreatic exocrine secretion. However, the potential effects of ghrelin on the morphology of exocrine pancreas (EP) remain unknown. In this work, using fractal analysis, we demonstrate that centrally administered ghrelin increases structural complexity and tissue disorder in rat EP. The study was carried out on a total of 40 male Wistar rats divided into four groups (n = 10): ghrelin-treated animals (average age, 1.5 months), ghrelin-treated animals (8.5 months), and controls (1.5 and 8.5 months). The pancreas tissue sections were stained with hematoxylin/eosin and visualized by light microscopy. For each animal, the average values of tissue fractal dimension, lacunarity, as well as parameters of co-occurrence matrix texture, were determined using tissue digital micrographs. The results indicate that ghrelin administration increases EP fractal dimension and textural entropy, and decreases lacunarity, regardless of the age. To our knowledge, this is the first study to investigate the effects of ghrelin on the morphological properties of pancreatic tissue, and also the first to apply fractal and textural analysis methods in quantification of EP tissue architecture.

Central ghrelin treatment stimulates ACTH cells in normal-fed, food-restricted and high-fed rats: An immunohistomorphometric and hormonal study.

Changes in feeding regime represent serious stress, while ghrelin is considered a key player in energy balance. We investigated the effects of intracerebroventricular (ICV) ghrelin application on pituitary adrenocorticotropic (ACTH) cells in rats fed diets differing in energy content. Before the ICV treatment, male Wistar rats were subjected to three different feeding regimes for 4 weeks: normal-fed (NF), food-restricted (FR) or high-fed (HF) (n = 3 × 14). At the age of 8 weeks, rats from each group were divided into two subgroups and given ICV, either ghrelin (G; 1 µg ghrelin/5 µl PBS, n = 7) or solvent alone (5 µl PBS, n = 7) every 24 h for 5 days. The immunohistochemical appearance and quantitative morphology of pituitary ACTH cells were evaluated, as well as peripheral ACTH and corticosterone levels. Central ghrelin administration increased (p<0.05) ACTH cell volumes in GNF, GFR and GHF rats by 8.1%, 11.8% and 9.1%, respectively, compared to the controls, while significant increases in ACTH cell volume density were observed in GNF and GHF rats. Circulating ACTH and corticosterone levels were elevated (p<0.05) in GNF and GFR rats by 72.8% and 80.8%, respectively, when compared to the corresponding controls. Thus, central ghrelin administration stimulated the pituitary-adrenal axis under preserved and negative energy balance states.

Age-dependent modulation of central ghrelin effects on food intake and lipid metabolism in rats.

Ghrelin is an endogenous peptide potentially useful in therapy of anorexia and other age-related metabolic disturbances. We evaluated the influence of age on the orexigenic and lipid metabolism-altering effects of ghrelin. Peripubertal, young, adult and middle-aged rats (1, 2, 7 and 12 months old, respectively) were treated with 5 daily intracerebroventricular injections of ghrelin (0.15 nmol) or saline (control). The food intake was measured daily before treatment, while white adipose tissue and serum/plasma samples for detection of lipid metabolites/hormones were collected at the end of the experiment. The values of cumulative food intake and body weight gain declined, while the white adipose tissue deposits and blood concentrations of triglycerides, cholesterol and free fatty acids all increased with age. Ghrelin significantly increased all parameters, but the stimulatory effects on body weight gain and food intake were more pronounced in peripubertal/young rats, while the increase in white adipose mass, triglyceride and low-density lipoprotein cholesterol levels was more noticeable in adult/middle-aged animals. The decrease in sensitivity to ghrelin-mediated stimulation of food intake in older animals could not be explained by alterations in ghrelin's ability to reduce anorexigenic hormones insulin and leptin. However, the higher responsiveness of aged rats to ghrelin-mediated increase in lipid metabolites was accompanied by an increase in adrenocorticotropic hormone and corticosterone levels. These results indicate that aging, while reducing sensitivity to ghrelin-mediated increase in body weight gain and food intake, might enhance the responsiveness to the stimulatory effects of ghrelin on lipid metabolites and hypothalamic-pituitary-adrenal axis activity.

Complexity reduction of chromatin architecture in macula densa cells during mouse postnatal development.

AIM: To determine whether complexity of chromatin structure in kidney macula densa cells (MDC) decreases during postnatal development in mice. METHODS: The levels of chromatin structural complexity were measured by determining fractal dimension of MDC nuclei. Kidney tissue was obtained from the total of 32 male Swiss albino mice divided into four age groups (n = 8): newborn (0 days), 10 days old, 20 days old and 30 days old. For a total of 640 MDC chromatin structures, fractal dimension, lacunarity, as well as parameters of Grey level co-occurrence matrix (GLCM) texture were determined. RESULTS: Chromatin fractal dimension in animals aged 10 days, 20 days and 30 days was significantly lower (P < 0.05, P < 0.01 and P < 0.001, respectively), compared with newborn mice. This complexity reduction of chromatin architecture is in accordance with previously published studies, which detected generalized and sustained loss of both tissue and cell complexity during aging. The loss of complexity was texture-independent, since there was no statistically significant difference (P > 0.05) in both chromatin angular second moment and inverse difference moment between the age groups. CONCLUSION: Our results indicate that age-related nuclear intrinsic factors which do not influence chromatin texture may have an important role in MDC postnatal development.

Histomorphometric features of ventral prostate in different aged rats after central ghrelin treatment.

Ghrelin, the endogenous ligand of growth hormone secretagogue receptor type 1a (GHS-R1a), has emerged as pleiotropic modulator of diverse biological functions, including energy homeostasis and recently, reproduction. The influence of intracerebroventricularly (ICV) administered ghrelin (1 µg/day/rat for 5 days) to rats of different ages, i.e, peripubertal (38 days), adult (60 days) and middle-aged (180 days) on the ventral prostate size and morphology, serum testosterone levels and testis weight was examined. Ghrelin treatment significantly increased (p < 0.05) absolute ventral prostate weight in peripubertal and middle-aged rats, by 27% and 37% respectively, due to enhancement of epithelial and/or luminal compartment of the gland. In adult rats, both absolute and relative volumes of the acinar lumen were significantly decreased (p < 0.05), by 38% and 44% respectively, which was associated with significant increases (p < 0.05) in relative and absolute volumes of interacinar stroma, whereas ventral prostate weigh was unchanged. Irrespective of animal age, ghrelin did not affect serum testosterone levels. These are the first results of ghrelin treatment effects on healthy prostate appearance, which allow us to conclude that the rat ventral prostate response to ghrelin depends on the developmental stage of animals. Our results merit further investigations and may have clinical implications, especially in the light of data on possible role of ghrelin in prostate hypertrophy and adenomas.

Intracerebroventricular administration of metformin inhibits ghrelin-induced Hypothalamic AMP-kinase signalling and food intake.

BACKGROUND/AIMS: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. METHODS: Rats were injected intracerebroventricularly with ghrelin (5 µg), metformin (50, 100 or 200 µg), 5-amino-imidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR, 25 µg) and L-leucine (1 µg) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. RESULTS: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, S6K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting S6K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. CONCLUSION: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus.

Immunomodulatory actions of central ghrelin in diet-induced energy imbalance.

We investigated the effects of centrally administered orexigenic hormone ghrelin on energy imbalance-induced inflammation. Rats were subjected for four weeks to three different dietary regimes: normal (standard food), high-fat (standard food with 30% lard) or food-restricted (70%, 50%, 40% and 40% of the expected food intake in 1st, 2nd, 3rd and 4th week, respectively). Compared to normal-weight controls, starved, but not obese rats had significantly higher levels of proinflammatory cytokines (TNF, IL-1ß, IFN-γ) in the blood. When compared to normally fed animals, the hearts of starved and obese animals expressed higher levels of mRNAs encoding proinflammatory mediators (TNF, IL-1ß, IL-6, IFN-γ, IL-17, IL-12, iNOS), while mRNA levels of the anti-inflammatory TGF-ß remained unchanged. Intracerebroventricular (ICV) injection of ghrelin (1 µg/day) for five consecutive days significantly reduced TNF, IL-1ß and IFN-γ levels in the blood of starved rats, as well as TNF, IL-17 and IL-12p40 mRNA expression in the hearts of obese rats. Conversely, ICV ghrelin increased the levels of IFN-γ, IL-17, IL-12p35 and IL-12p40 mRNA in the heart tissue of food-restricted animals. This was associated with an increase of immunosuppressive ACTH/corticosterone production in starved animals and a decrease of the immunostimulatory adipokine leptin both in food-restricted and high-fat groups. Ghrelin activated the energy sensor AMP-activated protein kinase (AMPK) in the hypothalamus and inhibited extracellular signal-regulated kinase (ERK) in the hearts of obese, but not starved rats. Therefore, central ghrelin may play a complex role in energy imbalance-induced inflammation by modulating HPA axis, leptin and AMPK/ERK signaling pathways.

Central effects of ghrelin on the adrenal cortex: a morphological and hormonal study.

Ghrelin, a growth hormone secretagogue that exerts an important role in appetite and weight regulation, participates in the activation of the hypothalamo-pituitary-adrenal (HPA) axis. Male Wistar rats (5/group) received daily for 5 days, via an ICV (intracerebroventricular) cannula, 5 microl phosphate buffered saline with or without 1 microg of rat ghrelin. Two hours after the last injection, blood and adrenal glands were collected from decapitated rats for blood hormone analyses and histologic and morphometric processing. Ghrelin treatment resulted in increased (p<0.05) body weight (13%), absolute whole adrenal gland weight (18%) and whole adrenal gland volume (20%). The absolute volumes of the entire adrenal cortex, ZG, ZF, and ZR also increased (p<0.05) after ghrelin by 20%, 21%, 21% and 11%, respectively. Ghrelin-treated rats had elevated (p<0.05) blood concentrations of ACTH, aldosterone and corticosterone (68%, 32% and 67%, respectively). The data clearly provide both morphological and hormonal status that ghrelin acts centrally to exert a global stimulatory effect on the adrenal cortex. Clarifying of the ghrelin precise role in the multiple networks affecting the stress hormone release, besides its well known energy and metabolic unbalance effects, remains a very important research goal.

Overweight in trained subjects - are we looking at wrong numbers? (Body mass index compared with body fat percentage in estimating overweight in athletes.).

Body mass index (BMI) is widely used as an index of obesity in adults. In trained population, individual with low body fat could be classified as overweight by BMI. To evaluate this problem, the purposes of this study were to determine the BMI and body fat percentage (BF%) of trained and untrained subjects and to evaluate the accuracy of BMI classification (> or =25 kg.m(-2)) as a prediction of overweight/obesity in trained subjects. The total number of 299 trained (basketball players) and 179 untrained male subjects participated in this study. Body height and body mass were measured; BMI was calculated for all subjects. BF% was determined via Tanita bioimpedance body composition analyzer. BMI >or = 25 kg.m(-2) and BF% > 20% were used to define overweight. There was no significant age differences. Body mass, height (p < 0.01) and BMI (p < 0.05) were significantly higher, although BF% was significantly lower (p < 0.01) in trained group when compared to untrained. Eighty-five trained subjects had a BMI of 25 or higher, indicating overweight. Of these, only three individuls had excess BF%. The results of the present study suggest that a BMI > or = 25 kg.m(-2) is not an accurate predictor of overweight in trained subjects.

Central ghrelin affects pituitary-thyroid axis: histomorphological and hormonal study in rats.

Body weight depends on the balance between energy intake and consumption. An interaction between ghrelin and thyroid function has been reported only in pathophysiological states. We examined whether intracerebroventricular (ICV) administration of ghrelin affects the structure and function of the pituitary-thyroid axis in young adult male rats. Ghrelin (0.3 nmol/5 microl PBS) or an equal volume of PBS were injected every 24 h into the lateral cerebral ventricle for 5 days. Two hours after the last treatment the animals were killed, their pituitaries and thyroids excised and prepared for further histological, immunohistochemical and morphometric investigation. Serum TSH levels were measured by RIA, while the total T(4) and T(3) levels were examined by ECLIA. Ghrelin treatment increased pituitary weight (p < 0.05) when compared to the controls, with no effect on the thyroid weight. Smaller, degranulated TSH-immunopositive cells were noticed within the pituitaries of ghrelin-treated animals; their cellular and nuclear volume as well as the relative volume density of thyrotrophs decreased (p < 0.05) in comparison to the control values. The level of serum TSH was reduced (p < 0.05). In the thyroid parenchyma of ghrelin-treated rats, an increased number of hypofunctioning follicles was noticed, characterized by flattened, weakly Tg-immunoreactive epithelium and colloid distension. The relative volume densities of the follicles and colloid increased (p < 0.05), while the thyroid index of activation rate and the serum level of total T(4) decreased (p < 0.05). In conclusion, centrally applied ghrelin modulated the immunohistomorphometric features of pituitary TSH cells and decreased the level of serum TSH, consequently changing thyroid morphology and function, by reducing the T(4) hormone level in the serum.

[Initial 111In-platelet kinetics: indicator of platelet sequestration/destruction site or quality control of platelet labelling?].

Taking into consideration the existing disagreement in the literature, the aim of this paper was to estimate the value of the initial kinetics of autologous platelets labelled with 111In-oxinate, performed during the first 20 minutes after their intravenous injection. Two hypothesis were tested: 1. Initial 111In-platelet kinetics indicates the platelet sequestration site (in patients with normal mean platelet life span)/destruction site (in patients with shortened mean platelet life span), 2. Initial 111In-platelet kinetics indicates the quality of platelet separation and labelling procedure. We performed initial labelled platelet kinetics in thrombocytopenic patients (in order to test the first hypothesis) as well as in control (healthy) subjects (in order to test the second hypothesis). Thirty-nine persons were investigated: 33 with thrombocytopenia: 25 with shortened mean platelet life span, caused by chronic im mune thrombocytopenic purpura (ITP), eight with normal platelet life span and thrombocytopenia caused by myelodysplastic syndrome (MDS), six healthy, control subjects (C). In all 39 persons platelet blood count on the day of platelet labelling was determined, autologous platelet labelling with 111In-oxinate was performed, general and differential yields of platelet labelling (GYL and DYL), as well as mean labelled platelets life span were determined. Besides that, initial labelled platelets kinetics was performed with initial 111In-platelets accumulation in the liver (IPAL) calculation, as well as the late platelet kinetics for platelet sequestration index and platelet sequestration/destruction site determination. We obtained two types of initial labelled platelets kinetics (not only in the patients with shortened platelet life span, but also in the subjects with normal labelled platelets life span), which differed in the IPAL value and in the ratio of the liver and the heart radioactivity: IPAL < 20% and IPAL>20%. We found statistically significant difference in GYL and DYL between the two groups: IPAL<20% and IPAL > 20%. Both yields were higher in IPAL<20% group. There was no significant difference between the two IPAL groups in the platelet blood count, labelled platelet life span, sequestration index and sequestration site. No correlation could be found between IPAL on one side and platelet blood count, sequestration index, and sequestration site on another. We concluded that initial labelled platelet kinetics could not indicate the platelet sequestration/destructon site (which is accomplished by the late labelled platelets kinetics), but nevertheless, it is very sensitive and useful method of platelet separation and labelling quality control. While in vitro quality control parameters (GYL and DYL) indicate the quality of only one part of this procedure, initial labelled platelet kinetics reflects discrete platelet function disturbance that might happen from the moment of blood sample collection till the labelled platelets intravenous injection.

Somatostatin affects morphology and secretion of pituitary luteinizing hormone (LH) cells in male rats.

The somatostatin peptides (SRIH-14, SRIH-28) and their multiple receptors are generally associated with anti-proliferative and anti-secretory actions. This study compared, using standard morphometric measurements and terminal serum LH concentrations, effects of intracerebroventricular (icv) SRIH-14 and SRIH-28 in nanomolar amounts on immunohistochemically identified LH cells in pituitary glands of male rats. Rats received l microg/5 microl of SRIH-14 or SRIH-28 icv on days 1,3, and 5, whereas control rats received only icv saline. Animals were killed 5 days later for serum LH assays. Pituitarys were harvested for PAP immunohistochemistry and morphometry. Morphometric measurements were made by an observer blinded to the treatment group. Histochemically identified LH cells from both SRIH groups appeared smaller, often pycnotic and darkly stained compared to those from saline-treated rats. Both SRIH treatments reduced (p < 0.05) the quantitative morphometric measurements for cell volume, nuclear volume, and relative volume density. Both SRIH treatments also reduced serum LH concentration (p < 0.05), supporting the hypothesis that systemic physiology was altered. Collectively, the data support the opinion that nanomolar amounts of either SRIH peptide, acting on receptors reached from cerebrospinal fluid, exert an anti-secretory effect on LH cells of male rats. Modifications of central SRIH receptors may provide an approach for treatment of male sexual dysfunction and/or be of pathophysiologic significance in these disturbances.