Influence of amygdala catecholamines on ovarian and adrenal medullary secretion.

The amygdaloid complex participates in the modulation of endocrine functions, and contains measurable amounts of noradrenaline (NA) and dopamine (DA). This study examined the contribution of the amygdaloid catecholaminergic systems to the regulation of the adrenal medulla and the ovary. To accomplish this the neurotoxin 6-hydroxydopamine (6-OHDA) was bilaterally injected into the basolateral nucleus of the amygdala (ABL) in cycling rats. The contents of NA and DA in right and left amygdala decreased significantly in lesioned animals with respect to sham lesioned animals, but hypothalamic levels were not different between groups. Administration of 6-OHDA to rats increased the NA, DA and adrenaline (A) contents of the adrenals compared to vehicle treated rats. In addition, lesioned animals showed a significant increase of NA and DA contents in the ovary, although A levels did not differ between groups. Serum oestradiol (O) concentrations were significantly lower in lesioned animals than in controls. These data suggest that the amygdaloid catecholaminergic systems exert an inhibitory effect on catecholamine content of the adrenals and the ovary, and influence the ovarian oestradiol secretion mechanism.     

Flux of catecholamines through chromaffin vesicles in cultured bovine adrenal medullary cells

Primary cultures of bovine adrenal medullary chromaffin cells were pulse-labeled with [3H]dopamine or [3H]norepinephrine and examined for radioactive and total catecholamine contents by high performance liquid chromatography after additional incubations of 15 min to 10 days. [3H]Dopamine was rapidly taken up by chromaffin vesicles in situ and converted to norepinephrine with a half-time of approximately 6 h. [3H] Norepinephrine taken up by the cells was metabolized in three phases. 1) During its brief transit through the cytoplasm, 20 to 35% of this amine was converted to [3H]epinephrine. 2) Following vesicular accumulation, 65 to 70% of the remaining [3H]norepinephrine was methylated to form [3H]epinephrine with a half-time of approximately 30 h, corresponding to the rate of vesicular catecholamine loss from reserpine-treated cells. 3) The residual [3H]norepinephrine decreased with a half-time of 5 days, probably representing loss from norepinephrine-storing cells. [3H]Epinephrine formed endogenously had a half-life in the cultures of approximately 15 days. These data suggest that leakage of norepinephrine from chromaffin vesicles into the cytoplasm limits the rate of dopamine conversion to epinephrine in the adrenal medulla. The kinetic data indicate that approximately 18% of the endogenous norepinephrine and 73% of the endogenous dopamine are present in epinephrine cells.     

Sympathetic innervation of white adipose tissue and its regulation of fat cell number

White adipose tissue (WAT) is innervated by the sympathetic nervous system (SNS), and the central origins of this innervation have been demonstrated for inguinal and epididymal WAT (iWAT and eWAT, respectively) using a viral transneuronal tract tracer, the pseudorabies virus (PRV). Although the more established role of this sympathetic innervation of WAT is as a major stimulator of lipid mobilization, this innervation also inhibits WAT fat cell number (FCN); thus, local denervation of WAT leads to marked increases in WAT mass and FCN. The purpose of this study was to extend our understanding of the SNS regulation of FCN using neuroanatomical and functional analyses. Therefore, we injected PRV into retroperitoneal WAT (rWAT) to compare the SNS outflow to this pad from what already is known for iWAT and eWAT. In addition, we tested the ability of local unilateral denervation of rWAT or iWAT to promote increases in WAT mass and FCN vs. their contralateral neurally intact counterparts. Although the overall pattern of innervation was more similar than different for rWAT vs. iWAT or eWAT, its SNS outflow appeared to involve more neurons in the suprachiasmatic and solitary tract nuclei. Denervation produced significant increases in WAT mass and FCN for both iWAT and rWAT, but FCN was increased significantly more in iWAT than in rWAT. These data suggest differences in origins of the sympathetic outflow to WAT and functional differences in the WAT SNS innervation that could contribute to the differential propensity for fat cell proliferation across WAT depots in vivo.     

Sympathetic denervation of one white fat depot changes norepinephrine content and turnover in intact white and brown fat depots

It is well-established that the sympathetic nervous system (SNS) regulates adipocyte metabolism and recently it has been reported that sensory afferents from white fat overlap anatomically with sympathetic efferents to white fat. The studies described here characterize the response of intact fat pads to selective sympathectomy (local 6-hydroxydopamine (6OHDA) injections) of inguinal (ING) or epididymal (EPI) fat in male NIH Swiss mice and provide in vivo evidence for communication between individual white and brown fat depots. The contralateral ING pad, both EPI pads, perirenal (PR), and mesenteric (MES) pads were significantly enlarged 4 weeks after denervating one ING pad, but only intrascapular brown adipose tissue (IBAT) increased when both ING pads were denervated. Denervation of one or both EPI pad had no effect on fat depot weights. In an additional experiment, norepinephrine turnover (NETO) was inhibited in ING, retroperitoneal (RP), MES, and IBAT 2 days after denervation of both EPI or of both ING pads. NE content was reduced to 10-30% of control values in all fat depots. There was no relation between early changes in NETO and fat pad weight 4 weeks after denervation, even though the reduction in NE content of intact fat pads was maintained. These data demonstrate that there is communication among individual fat pads, presumably through central integration of activity of sensory afferent and sympathetic efferent fibers, that changes sympathetic drive to white adipose tissue in a unified manner. In specific situations, removal of sympathetic efferents to one pad induces a compensatory enlargement of other intact depots.     

Quantal secretion of catecholamines measured from individual bovine adrenal medullary cells permeabilized with digitonin.

Secretion of catecholamines from individual bovine adrenal medullary cells grown in primary culture has been investigated with a carbon-fiber microelectrode placed adjacent to the cells. Oxidation of catecholamines at the electrode surface results in changes in current, which give a real-time measure of catecholamine secretion. Chemical agents are introduced to the individual cells by pressure ejection from micropipettes. When incubated in Ca(2+)-containing buffers, secretion is not observed. However, permeabilization of the cell by exposure to 20 microM digitonin for approximately 15 s results in a Ca(2+)-dependent secretion, and the contents of individual vesicles are detected in the form of sharp spikes. The rate at which spikes occur is a function of the Ca2+ concentration in the external media and reaches a maximum at 19 microM Ca2+. The area of the spikes range from 0.1 to greater than 10 picocoulombs, but the majority are less than 2 picocoulombs, corresponding to less than 6 x 10(6) molecules detected per spike. Histograms of the spike areas are essentially independent of the Ca2+ concentration, indicating that the population of vesicles which undergo exocytosis is the same for all concentrations. Exocytotic secretion can be distinguished from nonexocytotic release by analysis of the shape of the spikes.     

Time course of release of catecholamines from individual vesicles during exocytosis at adrenal medullary cells.

The time course of extrusion of the vesicular contents during exocytosis has been examined at adrenal medullary cells with carbon-fiber microelectrodes. Two electrochemical techniques were used: cyclic voltammetry and amperometry. Spikes obtained by amperometry had a faster time course than those measured by cyclic voltammetry, consistent with the different concentration profiles established by each technique. However, the experimental data obtained with both techniques were temporally broadened with respect to dispersion of an instantaneous point source by diffusion. Measurements with the electrode firmly pressed against the cell surface established that the temporal broadening is a result of a rate-limiting kinetic step associated with extrusion of the vesicular contents at the cell surface. The data do not support a rate-limiting process due to restricted efflux from a small pore. When combined with previous results, the data suggest that the rate-limiting step for chemical secretion from adrenal medullary cells during exocytosis is the dissociation of catecholamines from the vesicular matrix at the surface of the cell.     

Developmental changes in fat body and midgut chromosomes of Drosophila auraria

Changes in puffing activity of fat body (FB) and midgut (MG) chromosomes of Drosophila auraria during late larval and white prepupal development as well as after in vitro culture with or without ecdysterone were studied and compared with those of the salivary gland (SG). The Balbiani Rings characteristic of the SG chromosomes of D. auraria, are not formed in FB and MG. Most of the inverted tandem chromosomal duplications that have been found to be common to all three tissues showed differentiation of puffing activity of the bands considered to be homologous. The major early ecdysone puffs 73A and 73B (considered to be homologues of D. melanogaster puffs 74EF and 75B, respectively), together with other early ecdysone puffs were present in all three tissues. Clear intermoult and postintermoult puffs were not evident in FB and MG chromosomes. However, a small set of late ecdysone puffs could be scored in FB, while no late ecdysone puffs were abserved in MG. Other tissue-specific puffs were identified, but a very small number of them were limited to MG.by W. Beermann     

Activity-related changes of body fat and motor performance in obese seven-year-old boys

The effects of 35 weeks of extra-curricular, mainly aerobic, dynamic physical activity were analysed in overweight and obese 7-year-old boys contrasted with control groups. Body composition was estimated by using the body mass index (BMI) and skinfold thicknesses. Overweight or obesity was defined according to the suggestions of Cole and associates (2000). The activity program consisted of swimming and water games, folk dance, and soccer. Data were collected four times between September 2003 and October 2004. Thirty-one overnight or obese boys volunteered to participate in the activity program (weekly, to physical classes of 45 min. plus three extra-curricular activity sessions of 60 min. duration). The control subjects were 43 overweight or obese boys, and 75 non-overweight and non-obese ones. The controls had only two curricular physical education classes every week. Physical performance capacity was tested by 30 m dash, 400 m run, standing long jump, and fist-ball throw. Body fat content estimated by taking the sum of five skinfolds decreased significantly during the 35-week training program. However, body weight as well as skinfold thicknesses increased significantly during the four month non-active period that followed. Physical performance improved during the test period, but deteriorated between the third and fourth data collections. BMI, as well as the sum of five skinfolds increased in both control groups.Physical performance decreased in the overweight control subjects and increased moderately in the non obese ones. We inferred that more vigorous habitual exercise alone, i.e., without a program of dietary control, though effective, could not efficiently stabilise body fat, still less achieve a lasting reduction of it. Obese, but also overweight subjects need long term exercise programs of sufficient intensity, duration and frequency, plus dietary measures, to get rid of excess body fat.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.     

Localization and characterization of carbohydrates in adrenal medullary cells

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.     

Morpho-functional changes of fat body in bacteria fed Drosophila melanogaster strains

We have examined the addition of Escherichia coli to the diet at day 0 of adult life of females from two Oregon R Drosophila melanogaster strains, selected for different longevities: a short-life with an average adult life span of 10 days and a long-life standard R strain with an average adult life span of 50 days. The addition of bacteria to the diet significantly prolonged the fly longevity in both strains and affected the structure and histochemical reactivity of the fat body. The increased survival was characterized by great amount of glycogen accumulated in fat body cells from both strains. In aged control animals, fed with standard diet, lipid droplets were seen to be stored in fat body of short-lived, but not long-lived, flies. On the whole, our data indicate that exogenous bacteria are able to extend the survival of Drosophila females, and suggest that such a beneficial effect can be mediated, at least in part, by the fat body cells that likely play a role in modulating the accumulation and mobilization of reserve stores to ensure lifelong energy homeostasis.     

Gender specific correlations of adrenal gland size and body fat distribution: a whole body MRI study.

OBJECTIVE: The aim of this study was to investigate the gender specific correlations of stress related tissues [adrenal gland volume (AV), visceral fat] and alimentary dependent fat compartments with cortisol concentrations in healthy male and female subjects. METHODS: Fourteen men and 13 women were examined. Fat compartments [whole body fat, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SCAT)] were determined using whole body MRI. Adrenal gland volume was assessed by a 3D MR data set. The salivary cortisol was determined at 9 AM and 4 PM. RESULTS: Men had significantly more visceral fat and less subcutaneous fat than women. Adrenal gland size correlated significantly with the visceral and subcutaneous fat in women (r=0.7, p=0.008), but not in men (r=0.2, p=0.4). There was a negative correlation between the decrease of cortisol between 9 AM and 4 PM with VAT (r=-0.451, p=0.027) in the whole group. DISCUSSION: The high correlation between the adrenal gland volume and VAT in women underlines the link between hypothalamic-pituitary-adrenal (HPA) axis, stress, and circadian cortisol rhythm, respectively, and an increased abdominal fat volume. The lack of correlation between visceral fat and adrenal volume in men points to an additional influence of sex hormones.