Role of calcium in the secretion of leptin from white adipocytes

The mechanism by which calcium regulates leptin secretion was studied in adipocytes isolated from rat white adipose tissue. Incubation of adipocytes in a medium containing glucose, but no calcium, markedly inhibited insulin-stimulated leptin secretion (ISLS) and synthesis, without affecting basal leptin secretion or lipolysis. However, when pyruvate was used as a substrate, ISLS was insensitive to the absence of calcium. Likewise, the stimulatory effects of insulin were completely prevented by phloretin, cytochalasin B, and W-13 (3 agents that interfere with early steps of glucose metabolism) in the presence of glucose, but not in the presence of pyruvate. Thus calcium appears to be specifically required for glucose utilization. On the other hand, (45)Ca uptake and leptin secretion were not affected by insulin or by inhibitors of L-type calcium channels. However, agents increasing plasma membrane permeability to calcium (high calcium concentrations, A-23187, and ATP) increased (45)Ca uptake and concomitantly inhibited ISLS. Similarly, release of endogenous calcium stores by thapsigargin inhibited ISLS in a dose-dependent manner. ATP, A-23187, calcium, and thapsigargin inhibited ISLS, even in the presence of pyruvate. These results show that 1) extracellular calcium is necessary for ISLS, mainly by affecting glucose uptake, 2) insulin does not affect extracellular calcium uptake, and 3) increasing cytosolic calcium by stimulating its uptake or its release from endogenous stores inhibits ISLS at a level independent of glucose metabolism. Thus calcium regulates leptin secretion from adipocytes in a manner that is markedly different from its role in the exocytosis of many other polypeptidic hormones.     

Regulation of leptin secretion from white adipocytes by free fatty acids

Norepinephrine stimulates lipolysis and concurrently inhibits insulin-stimulated leptin secretion from white adipocytes. To assess whether there is a cause-effect relationship between these two metabolic events, the effects of fatty acids were investigated in isolated rat adipocytes incubated in buffer containing low (0.1%) and high (4%) albumin concentrations. Palmitic acid (1 mM) mimicked the inhibitory effects of norepinephrine (1 microM) on insulin (10 nM)-stimulated leptin secretion, but only at low albumin concentrations. Studies investigating the effects of the chain length of saturated fatty acids [from butyric (C4) to stearic (C18) acids] revealed that only fatty acids with a chain length superior or equal to eight carbons effectively inhibited insulin-stimulated leptin secretion. Long-chain mono- and polyunsaturated fatty acids constitutively present in adipocyte triglyceride stores (oleic, linoleic, gamma-linolenic, palmitoleic, eicosapentanoic, and docosahexanoic acids) also completely suppressed leptin secretion. Saturated and unsaturated fatty acids inhibited insulin-stimulated leptin secretion with the same potency and without any significant effect on basal secretion. On the other hand, inhibitors of mitochondrial fatty acid oxidation (palmoxirate, 2-bromopalmitate, 2-bromocaproate) attenuated the stimulatory effects of insulin on leptin release without reversing the effects of fatty acids or norepinephrine, suggesting that fatty acids do not need to be oxidized by the mitochondria to inhibit leptin release. These results demonstrate that long-chain fatty acids mimic the effects of norepinephrine on leptin secretion and suggest that they may play a regulatory role as messengers between stimulation of lipolysis by norepinephrine and inhibition of leptin secretion.     

Expression of an Antigen Associated with Basal Bodies of Human Ciliated Epithelial Cells

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000–45,000Mr protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9+0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.     

Sex differences in adrenocortical structure and function

Summary The zonation and cellular composition of the adrenal cortex of intact mature male and female rats of different strains (Chbb Thomm, CFY) and three strains of Wistar rats (W1, W2 and W3) at the age of 84–90 days were compared using morphometry.Both absolute and relative adrenal gland weights were higher in female than male rats. Rats of W3 and Chbb Thomm strains had the largest adrenals. These differences depended upon the higher volume of the zona fasciculata (ZF) and zona reticularis (ZR) in the W3 and Chbb Thomm strains than in the remaining animals. Females had larger adrenocortical zones than corresponding males. The average volume of the ZF cell ranged in males from 1589 m³ (W1 strain) to 2111 m³ (Chbb Thomm) and in females from 2249 m³ (W1 strain) to 2894 m³ (W3 strain). The average nuclear volume of the ZF cells was higher in female rats whereas there were no strain-and sex-differences in the size of zona glomerulosa (ZG) and ZR cells.The total number of parenchymal cells per pair of adrenals varied markedly among the studied strains, with the highest number in W3 and Chbb Thomm females. W3, Chbb Thomm and CFY females had larger number of parenchymal cells than males; the reverse was true for W1 strain whereas no differences were found among W2 male and female rats.     

Gestational changes in hamster adrenal cortex: Morphometric and ultrastructural stereologic studies

Summary In the hamster, the weight of the adrenal glands increases during the course of gestation, with the highest value at day 5. In comparison to non-pregnant control animals, there were no changes in the volume of the zona glomerulosa (ZG) and zona fasciculata (ZF), while the volume of the zona reticularis (ZR) increased notably. The average volume of ZG-cells rose at day 5 of pregnancy and thereafter gradually decreased to that of control hamsters. A marked drop in the volume of ZF-cells was seen at days 5 and 10 of pregnancy, whereas at day 15 the cells were larger than in controls. At day 5 of pregnancy, a conspicuous increase in the cell volume was found in ZR, followed by lower values at day 10 and again higher than in control hamsters at day 15. The total number of parenchymal cells in hamster adrenal cortex increased at day 10 of gestation, then underwent a marked decrease, reaching the control value at the final day of pregnancy; this drop was mainly due to a reduction in the number of ZF-cells. The changes in the cell volume were paralleled by rather proportional changes in the volume of the mitochondrial compartment and in the quantity of smooth endoplasmic reticulum. The volume of the lipid-droplet compartment significantly rose in the course of gestation in both ZF- and ZR-cells. The cortisol output by adrenal homogenates gradually decreased during pregnancy.Study performed in partial fulfillment of the Ph.D. Thesis of Magdalena Nowak     

The effects of ageing on the morphology and function of the zonae fasciculata and reticularis of the rat adrenal cortex

Summary The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.     

Morphology and evolution of the nervous system in Gnathostomulida (Gnathifera,Spiralia)

Within Spiralia, Gnathifera may represent the deepest branching lineage comprising the jaw worms Gnathostomulida and their sister group Micrognathozoa + Syndermata. Yet, very few nervous system studies have been conducted on this lineage of microscopic, jaw-bearing worms, limiting our understanding of the evolution of this organ system in Spiralia. The nervous system of representatives from all major groups of Gnathostomulida was here mapped using confocal laser scanning microscopy and immunohistochemistry. Their intra-epidermal, unsegmented nervous systems comprise an anterior brain and three to five ventral and two to four dorsal longitudinal nerves, connected by few transverse commissures. Neurites of the stomatogastric nervous system were found lining the pharynx and connecting to a prominent buccal ganglion. Supposedly, sensory ciliated cells in the pharynx and the gut were documented for the first time. Based on these morphological results, primary homologies of neural structures in Gnathostomulida and other Gnathifera were hypothesized and thereafter tested using parsimony. This first neurophylogeny of Gnathostomulida resulted in a topology congruent with molecular data, supporting the monophyly of Bursovaginoidea, Conophoralia, and Scleroperalia. From this topology, the evolution of the gnathostomulid nervous system was reconstructed. It suggests a specialization and diversification of cords and serotonin-like immunoreactive cell patterns from a plesiomorphic neuroarchitecture of three unsegmented nerve cords and a compact anterior brain and buccal ganglion. These plesiomorphic states resemble the nervous system of Micrognathozoa, and possibly the ancestral states of Spiralia.