The etiology of obesity involves imbalanced energy intake and utilization. ASP is an adipose tissue hormone that facilitates adipocyte uptake of serum fatty acids and their storage. Mice lacking ASP have less adipose tissue mass, despite increased food intake, than wild-type littermates. We hypothesize that the unstored fuels are oxidized through UCP (thermogenic mitochondrial carriers). / In male ASP-deficient mice mRNA levels were measured by semi-quantitative RT-PCR and the following changes were observed: UCP-1 decreased in all tested tissues, UCP-2 increased by 15% and 6 fold in muscle and white adipose tissue and UCP-3 increased 2.5 and 10 fold in muscle and epididymal adipose tissue, respectively. In female ASP-deficient mice UCP-1 decreased in all tissues, UCP-2 increased by 10% and 40% in inguinal and brown adipose tissue, respectively, and UCP-3 remained stable in all tissues. High fat diet nullified these differences, and decreased all wild-type UCP levels. / We propose that UCP-2 and 3 assume the role of UCP-1 in fuel utilization, thus helping mice face an increased energy load in the absence of ASP.

Acute administration of estrogen results in the vasodilatation and in the release of nitric oxide (NO) that occurs through activation of the serine-threonine kinase Akt/protein-kinase-B (PKB), which is known to increase the eNOS activity. 10-8 M of 17-beta-estradiol resulted in a left shift of the vasodilatory response to Ach in preconstricted aortic rings from oophorectomized rats (EC50 = 0.7 x 10-8 M with 17-beta-estradiol and 0.15 x 10-7 M of Ach without 17-beta-estradiol, P < 0.05). The effect was blocked by pre-treatment with Wortmannin, a PI(3)K inhibitor. AKT/PKB was phosphorylated in endothelial cells (EC) as early as 1-minute after estradiol-stimulation. Phosphorylation of eNOS and NO release in EC treated with 17-beta-estradiol were also increased. We conclude that the AKT/PKB pathway is involved in the acute release of NO by estrogen.

We examined the hypothesis that maturation ameloblasts degrade enamel matrix in a manner analogous to bone matrix degradation mediated by osteoclasts. Thus, we assessed the distribution of the cation-independent mannose 6-phosphate receptor (MRP) and lysosomal enzymes in the enamel organ and in the alveolar bone surrounding the rat incisor. The MPR was observed on the ruffled border of the ruffle-ended ameloblast (RA) but not on the distal call membrane of the smooth-ended ameloblast (SA), although both cells demonstrated strong immunoreactivity in the Golgi region. Immunogold localization of cathepsin B showed more labelling on the distal end of RA than SA, indicating that the source of the extracellular cathepsin B was likely the RA. Since MPR and lysosomal enzymes were also detected on the ruffled border of osteoclasts, our immunocytochemical approach provides strong evidence for a similarity between the maturation of enamel, as mediated by RAs, and bone matrix degradation by osteoclasts. / We also examined the nature of the basement membrane-like structure between maturation ameloblasts and the surface of enamel, and the possible role it may play in the maturation of enamel. We used high resolution electron microscopy combined with immunohistochemical localization of laminin, heparan sulfate proteoglycan (HSPG) and type IV collagen. Our results indicated that this structure was a highly specialized basement membrane unusually rich in HSPG. The cord network was replaced by a network of fine filaments, identified as core filaments of cords containing type IV collagen. A proposed role for this basement membrane is likely to be mediation of firm attachment of maturation ameloblasts to the surface of the enamel. / Since integrins play an essential role in cell-substratum interactions, we investigated the distribution of the integrin beta1 subunit on osteoclasts and ameloblasts. Immunocytochemistry showed significant concentrations of beta1 integrin at the ruffled border of osteoclasts, and negligible staining at the sealing zone. Since beta1 integrin localization was higher in the RA than the SA, we suggest that beta1 may have a role in the cell modulations. This role is likely to be recognition of extracellular matrix components and consequent interaction of RA with that matrix. The findings that the ruffled border of both RAs and osteoclasts display the most intense labelling for beta1 integrin, further supports our hypothesis that these cells are functionally similar.

Prosaposin is a multifunctional glycoprotein with different molecular masses and dual destinations. A 65 kDa form of prosaposin is targeted to lysosomes and converted by partial proteolysis, into four smaller non-enzymatic saposin A-D required for the hydrolysis of glycosphingolipids. However, the 65 kDa protein may be further glycosylated to a 70 kDa secretory form that is found in various biological fluids and suspected to have a trophic activity. Mutations of the prosaposin gene are linked to several lysosomal disorders. This thesis examines various aspects of the synthesis, targeting and function of prosaposin, and for practical purposes, the results and discussion were divided in three main sections. The first part deals with the cloning of the mouse prosaposin gene, the analysis of its transcribed mRNA and translation products. The second section examines the mechanism of targeting of the 65 kDa protein to lysosomes using mutagenic analyses. The third part deals with the effect of the inactivation of the prosaposin gene on the development of the male reproductive system. Sequence analysis revealed that the mouse prosaposin gene is over 20 kb in length and composed of 15 exons and 14 introns. Two forms of alternatively spliced mRNA (including or excluding exon 8) were found by RT-PCR in a tissue specific manner. Structure analysis and secondary structure predictions among mouse, rat and human prosaposins illustrated a common framework of amino acids forming amphiphatic helices enclosing an internal hydrophobic core implicated on their interaction with lipids. Mutagenic deletions of functional domains of prosaposin demonstrated that its C-terminus was required for the lysosomal targeting of this protein. Further evidence from chimeric constructs of albumin attached to various functional domains of prosaposin, suggested that the C-terminus plus at least one saposin domain are necessary for the targeting of albumin to lysosomes. Investigation of the effect of p

These investigations examined the relative function of costal and crural diaphragm segments. This work produced the first direct measurements of length and electromyogram (EMG) of the diaphragm in an awake, intact animal. / Examination of diaphragm function following laparotomy revealed a consistent pattern of postoperative segmental recovery, and showed the inadequacy of EMG alone as an indicator of diaphragm activity. Segmental contraction during airway occlusion was confirmed to be non-isometric and different per segment. The basic relation between segmental velocity of shortening and mean inspiratory flow, was confirmed for diaphragm but not for intercostal musculature. Anesthesia produced a distinctive alteration in the resting length of crural compared to costal segment, suggesting a difference in inherent segmental tonic activity. Costal and crural activity during hypoxic and hypercapnic stimulated breathing revealed different, stimulant-specific activities of the segments; hypoxia elicited prominent crural post inspiratory activity (PIIA). During thermal panting, peak crural shortening was out of phase with costal shortening and inspiratory airflow. This unique segmental asynchrony may represent a natural analog to high frequency ventilation. / We conclude that costal and crural diaphragm segments can function as individual segment-muscles, exhibiting distinctive, differential activities under certain conditions of respiration.

The liver is a major site of production of circulating levels of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs). We have used primary cultured rat hepatocytes maintained under serum free conditions to explore the regulatory role of various hormones on hepatic IGF-I and IGFBP-1 mRNA levels. / IGF-I mRNA levels were stimulated 2.0 to 2.5 fold by bovine growth hormone (bGH) and 1.8 to 2.0 fold by glucagon but on combining bGH and glucagon, a synergistic effect was observed and IGF-I mRNA level was augmented 10 to 12 fold. Octreotide blocked the hGH induced stimulation of IGF-I production in serum and hepatic IGF-I mRNA levels in hypophysectomized rats. This effect could have been partly due to the low levels of glucagon in serum when hypophysectomized rats were treated with hGH and octreotide. Octreotide was also found to inhibit GH stimulated IGF-I mRNA levels in rat hepatocytes. / The unique synergy observed with glucagon and bGH on IGF-I mRNA levels in hepatocytes was not reproduced by T$ sb3$, oPRL, dexamethasone, EGF or insulin when each was added in combination with bGH or glucagon. Like glucagon, the addition of IBMX or (Bu)$ sb2$cAMP stimulated IGF-I mRNA levels 1.8 to 2.0 fold, but in the presence of bGH, IGF-I mRNA levels were stimulated 10 to 12 fold. PMA stimulated IGF-I mRNA levels 1.2 to 1.4 fold but displayed no synergism when added with bGH. The stimulatory effect of bGH plus glucagon on IGF-I mRNA levels was inhibited in PKC depleted cells, in the presence of inhibitors of PKC and in the presence of cycloheximide. bGH had no posttranscriptional effect on IGF-I mRNA stability whereas glucagon or (Bu)$ sb2$cAMP stabilized IGF-I mRNA at a posttranscriptional level. / In summary, the major hormonal regulators of hepatic IGF-I mRNA levels appear to be GH and glucagon. Hepatic IGF-I mRNA levels are regulated by pathways involving protein kinase C and, protein kinase A as well as by synthesis of one or more protein(s). / Glucagon and dexamethasone each stimulated IGFBP-1 mRNA levels 3 to 4 fold whereas bGH and T$ sb3$ each inhibited IGFBP-1 mRNA levels 45 to 70%. Insulin, which inhibited IGFBP-1 mRNA levels 95%, was the most powerful inhibitor and was also found to inhibit IGFBP-1 mRNA levels in the presence of dexamethasone. IBMX and (Bu)$ sb2$cAMP stimulated IGFBP-1 mRNA levels 6 to 8 fold whereas PMA inhibited IGFBP-1 mRNA levels 40 to 50%. The inhibitory effect of bGH on IGFBP-1 mRNA levels was abolished in PKC depleted cells and also in the presence of inhibitors of PKC. In the presence of cycloheximide, IGFBP-1 mRNA was superinduced by bGH. bGH had no posttranscriptional effect on IGFBP-1 mRNA whereas glucagon and (Bu)$ sb2$cAMP stabilized IGFBP-1 mRNA at a postranscriptional level. / In summary, bGH, T$ sb3$ and insulin inhibited whereas dexamethasone and glucagon stimulated IGFBP-1 mRNA levels in hepatocytes. Effect of glucagon may be via elevation of cAMP levels, whereas the effect of bGH may be via activation of PKC levels. The inhibitory effect of bGH appears to require synthesis of one or more protein(s) besides stimulation of PKC levels.

This study investigates dyspnea and the mechanics of breathing during progressive exercise. Three subject groups, athletes, normal sedentary subjects and chronic obstructive diseased patients were studied during progressive exercise testing to exhaustion on a cycle ergometer. Subjects rated dyspnea on a Borg Scale. Inspiratory flow, esophageal/gastric pressures and rib cage/abdominal displacements were measured. / Subjects demonstrated two patterns of dyspnea response to changes in esophageal (pleural) pressure. All athletes, two normals and five patients were termed "low dyspnea responders", (LDR), whereas the remaining subjects were termed "high dyspnea responders", (HDR). / LDR demonstrated large, rapid negative gastric pressure swings, coupled with outward abdominal displacement during early inspiration when compared to HDR, suggesting that LDR utilized abdominal muscle relaxation at the onset of inspiration. This mechanism appears to provide an extra inspiratory force, contributing to the increasing pleural pressures required. This breathing pattern appears to diminish the sensation of dyspnea at a given pleural pressure.

The role of insulin-like growth factor I (IGF-I) in pancreatic islet cell growth and development has been debated in recent years. The dogma that IGF-I stimulates pancreatic islet growth has been challenged by combinational targeting of IGF or IGF-IR genes, as well as beta-cell-specific IGF-IR gene deficiency. In order to assess the physiological role of locally produced IGF-I, we have developed pancreatic-specific IGF-I gene deficiency (PID) by crossing Pdx1-Cre and IGF-I/loxP mice. PID mice were normal except for decreased blood glucose level and a 2.3-fold enlarged islet cell mass. When challenged with low doses of streptozotocin, control mice developed hyperglycemia after 6 days that was maintained at high levels for at least 2 months. In contrast, PID mice only exhibited marginal hyperglycemia after 12 days, maintained throughout the experiment. Furthermore, streptozotocin-induced beta-cell apoptosis (TUNEL assay) was significantly prevented in PID mice. PID mice also exhibited a delayed onset of type 2 diabetes induced by a high-fat diet, accompanied by super enlarged pancreatic islets and preserved sensitivity to insulin. As the phenotype is unlikely a direct consequence of IGF-I deficiency, we used oligonucleotide DNA microarray to explore possible activation of pro-islet genes in PID mice, which revealed upregulation of multiple new members of the Reg family genes (Reg2, 3alpha and 3beta) in the pancreas. The results were subsequently confirmed by Northern blot and/or realtime PCR, which exhibited 2 to 8 fold increases in the level of their mRNAs. Moreover, these Reg family genes were also activated following streptozotocin-induced beta-cell damage and diabetes. Our results reveal a possible mechanism of islet growth and protection in PID mice, thus serving a potential strategy in combating diabetes.

Three known mammalian endothelins, ET-1, ET-2, and ET-3, are each encoded by separate genes and expressed in a variety of vascular and nonvascular tissues. Two subtypes of endothelin receptors have been identified and termed endothelin-A and endothelin-B receptors (ET-A and ET-B). In the first part of this study, the ET-A gene was disrupted in mouse embryonic stem cells to generate mice deficient in ET-A. / In the second part of this study, we investigated a targeted disruption of the mouse ET-B gene that results in aganglionic megacolon associated with coat color spotting, resembling a hereditary syndrome of mice, humans and other mammalian species (Waardenburg syndrome). / In the third part of this study, we demonstrated that a targeted disruption of the mouse endothelin-3 ligand (ET-3) gene produces a similar recessive phenotype of megacolon and coat color spotting. / Two types of endothelin converting enzymes (ECE-1 and ECE-2) have been recently identified. In the last part of this study, we investigated the expression of ECE-1 in human tissues using immunohistochemistry and in situ hybridization, and compared it to those of ET-1 and big ET-1. (Abstract shortened by UMI.)

The time course of the spontaneous current spikes produced by the release of the catecholamines from individual vesicles was examined in bovine chromaffin cells by using the carbon filament technique in the amperometric mode. / Frequency histograms of the rise and decay times of the current spikes showed a paucity of very short duration events. Scatterograms of the rise and decay times consistently showed a positive relation, and the best fitted lines intercepted the ordinate (the axis of the decay time) at: 16.06 $ pm$ 6.45 msec (n = 11). / The effect of temperature changes upon the time course of release of content of individual vesicles in chromaffin cells was also examined. The amplitudes of the current spikes did not change significantly, whereas the rise times and the decay times diminished from (23.2 $ pm$ 11.6 to 11.9 $ pm$ 2.7 msec, and from 76.6 $ pm$ 25.4 to 47.3 $ pm$ 9.3 msec respectively) as the temperature was raised from 15$ sp circ$C to 35$ sp circ$C (n = 5). Nevertheless, the Q$ sb{10}$ values of the rise and decay times were surprisingly low. / The experimental findings suggest that in bovine chromaffin cells the duration of the release of content of single vesicles is much longer than in synapses. The results also suggest that this mechanism does not involve processes that are strongly temperature sensitive.