Ghrelin, a novel GH-releasing peptide isolated from human and rat stomach, stimulates food intake and GH secretion. We determined plasma ghrelin concentrations in patients with simple obesity, anorexia nervosa, and type 2 diabetes mellitus by RIA. We also studied plasma ghrelin responses to glucose load and meal intake and obtained a 24-h profile of circulating ghrelin in humans. Plasma ghrelin concentrations in patients with simple obesity and anorexia nervosa were lower and higher, respectively, than those of healthy subjects with normal body weight. Among those with type 2 diabetes mellitus, obese patients had lower and lean patients higher fasting plasma ghrelin concentrations than normal-weight patients. Fasting plasma ghrelin concentration was negatively correlated with body mass index in both nondiabetic and diabetic patients. Plasma ghrelin concentrations of normal subjects decreased significantly after oral and iv glucose administration; a similar response was also observed in diabetic patients after a meal tolerance test, reaching a nadir of 69% of the basal level after the meal. Circulating plasma ghrelin showed a diurnal pattern with preprandial increases, postprandial decreases, and a maximum peak at 0200 h. This study demonstrates that nutritional state is a determinant of plasma ghrelin in humans. Ghrelin secretion is up-regulated under conditions of negative energy balance and down-regulated in the setting of positive energy balance. These findings suggest the involvement of ghrelin in the regulation of feeding behavior and energy homeostasis.

ATP-sensitive K+ (KATP) channels play a crucial role in coupling metabolic energy to the membrane potential of cells. We have isolated a cDNA encoding a novel member (uKATP-1) of the inward rectifier K+ channel family from a rat pancreatic islet cDNA library. Rat uKATP-1 is a 424-amino acid residue protein (M(r) = 47,960). Electrophysiological studies of uKATP-1 expressed in Xenopus laevis oocytes show that uKATP-1 is a weak rectifier and is blocked with Ba2+ ions. Single-channel patch clamp study of clonal human kidney epithelial cells (HEK293) transfected with uKATP-1 cDNA reveals that uKATP-1 closes in response to 1 mM ATP and has a single channel conductance of 70 +/- 2 picosiemens (n = 6), indicating that uKATP-1 is an ATP-sensitive inward rectifier K+ channel. In addition, uKATP-1 is activated by the KATP channel opener, diazoxide. RNA blot analysis shows that uKATP-1 mRNA is expressed ubiquitously in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart, suggesting that uKATP-1 may play a physiological role as a link between the metabolic state and membrane K+ permeability of cells in almost every normal tissue. Since uKATP-1 shares only 43-46% amino acid identity with members of previously reported inward rectifier K+ channel subfamilies, including ROMK1, IRK1, GIRK1, and cKATP-1, uKATP-1 is not an isoform of these subfamilies and, therefore, represents a new subfamily of the inward rectifier K+ channel family having two transmembrane segments.

Xenopus oocytes indicates that calcium-activated chloride currents are evoked by PACAP and vasoactive intinal polypeptide, sggesting that PACAPR-3 can also be coupled to phospholipase C. RNA blot analysis studies reveal that PACAPR-3 mRNA is expressed at high levels in MIN6, at moderate levels in pancreatic islets and other insulin-secreting cell lines, HIT-T15 and RINm5F, as well as in the lung, brain, stomach, and colon, and at low levels in the heart. Furthermore, insulin secretion from MIN6 cells is scantiy stimulated by PACAP-38. These results suggest that the diverse biological effects of PACAP are mediated by a family of structurally related proteins and that PACAPR-3 participates in the regulation of insulin secretion.

Three isoforms of synaptotagmin, a synaptic vesicle protein involved in neurotransmitter release, have been characterized in the rat, although functional differences between these isoforms have not been reported. In situ hybridization was used to define the localization of synaptotagmin I, II, and III transcripts in the rat CNS and pituitary and adrenal glands. Each of the three synaptotagmin genes has a unique expression pattern. The synaptotagmin III gene is expressed in most neurons, but transcripts are much less abundant than the products of the synaptotagmin I and II genes. A majority of neurons in the forebrain expressed both synaptotagmin I and III mRNAs while synaptotagmin II gene expression was confined to subsets of neurons in layers IV-VI of the cerebral cortex, in the dentate granule cell region, the hilus, and the CA1-CA3 areas of the hippocampus. In the cerebellum, all three transcripts were visualized in the granule cell layer. Furthermore, synaptotagmin I probes revealed striking differences between distinct populations of neurons, as in addition to moderate labeling of granule cells, much more prominent hybridization signals were detected on scattered cell bodies likely to be Golgi interneurons. In the most caudal part of the brain, synaptotagmin II transcripts were abundant and were coexpressed with synaptotagmin III mRNAs. This pattern was found in putative motoneurons of the spinal cord, suggesting that the two isoforms might be involved in exocytosis at the neuromuscular junction. Only synaptotagmin I mRNAs were detected in the anterior and intermediate pituitary and in adrenal medullary cells. These data reveal an unexpectedly subtle segregation of the expression of synaptotagmin genes and the existence of multiple combinations of synaptotagmin isoforms which may provide diversity in the regulation of neurosecretion.

To clarify whether nicotine has a direct effect on the function of adipocytes, we evaluated nicotinic acetylcholine receptor (nAChR) expression in adipocytes by reverse transcriptasepolymerase chain reaction (RT-PCR) and immunocytochemistry and the direct effects of nicotine on the production of adipocytokines by enzyme-linked immunosorbent assay and Western blot analysis. Receptor binding assays were performed using [3 H]nicotine. RT-PCR studies revealed that ␣1-7, 9, 10, ␤1-4, ␦, and ⑀ subunit mRNAs are expressed in adipocytes. Immunocytochemical experiments also suggested the presence of ␣7 and ␤2 subunits. The receptor binding assay revealed a binding site for nicotine (K d ϭ 39.2 ϫ 10 Ϫ9