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Adipocytes contain large lipid droplets in their cytoplasm. When cultured, they float on top of the medium, clump together, and do not gain equal and sufficient access to the medium. Morphological changes cannot be observed and the majority of adipocytes undergo cell lysis within 72 h of isolation. We have used a ceiling culture method for human mature adipocytes which uses their buoyant property to allow them to adhere to a floating glass surface, where they remain viable for several weeks. Using confocal immunofluorescence microscopy we showed the cellular expression and subcellular localization of leptin in ceiling-cultured adipocytes. The secretion of leptin was increased from ceiling cultures following tumour necrosis factor-alpha treatment. Proliferation of mature human adipocytes in serum-containing medium was demonstrated by incorporation of bromodeoxyuridine, 2% of adipocytes showing positive incorporation after 4 h labelling. Proliferation was also evident from the budding of daughter cells. Apoptosis in the ceiling cultures was increased by 48 h serum deprivation (30-35 vs 10-15% in the control) and was assayed by propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling. Lipolysis, analysed by liquid scintillation counting, was increased by forskolin (10 microM for 90 min) and lipogenesis, shown by autoradiography, was stimulated by insulin (10 and 100 nM for 4 h). These findings indicate that ceiling-cultured adipocytes maintain adipocyte-specific functions and that ceiling culture, which overcomes the shortcomings of adipocyte suspension culture, can be used to study adipocyte cell biology.

Thyroid hormone (TH) deficiency results in delayed proliferation and migration of cerebellar granule cells. Although extensive cell loss during the development of the cerebellum under hypothyroid conditions is known, its nature and its mechanism are poorly understood. Bcl-2 family gene expression is known to determine the fate of cells to undergo apoptosis. We evaluated the effect of hypothyroidism on Bcl-2 family gene expression in the developing rat cerebellum. Electrophoresis and Western blotting were used to analyze DNA fragmentation and expression of DNA fragmentation factor (DFF-45), Bcl-2, Bcl-xL and Bax genes respectively. In the hypothyroid condition, extensive DNA fragmentation and enhanced cleavage of DFF-45 were seen throughout development (postnatal day 0 to day 24) and adulthood whereas they were absent in the euthyroid state. The anti-apoptotic genes Bcl-2 and Bcl-xL were down-regulated and the pro-apoptotic gene Bax was expressed at higher levels compared with the euthyroid state. These results suggest that normal levels of TH prevent cerebellar apoptosis to a large extent, whereas hypothyroidism not only increases the extent but also the duration of apoptosis by down-regulating the anti-apoptotic genes and maintaining a high level of the pro-apoptotic gene Bax.

Adipose tissue is an active endocrine organ, and our knowledge of this secretory tissue, in recent years, has led us to completely rethink how our body functions and becomes dysregulated with weight gain. Human adipose tissue appears to act as a multifunctional secretory organ with the capacity to control energy homoeostasis through peripheral and central regulation of energy homoeostasis. It also plays an important role in innate immunity. However, the capability to more than double its original mass to cope with positive energy balance in obesity leads to many pathogenic changes. These changes arise within the adipose tissue as well as inducing secondary detrimental effects on other organs like muscle and liver, including chronic low-grade inflammation mediated by adipocytokines (adipokine inflammation). This inflammation is modulated by dietary factors and nutrients including glucose and lipids, as well as gut bacteria in the form of endotoxin or LPS. The aim of this current review is to consider the impact of nutrients such as glucose and lipids on inflammatory pathways, specifically within adipose tissue. Furthermore, how nutrients such as these can influence adipokine inflammation and consequently insulin resistance directly through their effects on secretion of adipocytokines (TNFα, IL6 and resistin) as well as indirectly through increases in endotoxin is discussed.

Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly G_q- and to a lesser extent G_s-mediated; p38 activation even had a small G_i-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.