Effects of the obese gene product on body weight regulation in ob/ob mice.

C57BL/6J mice with a mutation in the obese (ob) gene are obese, diabetic, and exhibit reduced activity, metabolism, and body temperature. Daily intraperitoneal injection of these mice with recombinant OB protein lowered their body weight, percent body fat, food intake, and serum concentrations of glucose and insulin. In addition, metabolic rate, body temperature, and activity levels were increased by this treatment. None of these parameters was altered beyond the level observed in lean controls, suggesting that the OB protein normalized the metabolic status of the ob/ob mice. Lean animals injected with OB protein maintained a smaller weight loss throughout the 28-day study and showed no changes in any of the metabolic parameters. These data suggest that the OB protein regulates body weight and fat deposition through effects on metabolism and appetite.

The Stability Principle and global weak solutions of the free surface semi-geostrophic equations in geostrophic coordinates.

The semi-geostrophic equations are used widely in the modelling of large-scale atmospheric flows. In this note, we prove the global existence of weak solutions of the incompressible semi-geostrophic equations, in geostrophic coordinates, in a three-dimensional domain with a free upper boundary. The proof, based on an energy minimization argument originally inspired by the Stability Principle as studied by Cullen, Purser and others, uses optimal transport techniques as well as the analysis of Hamiltonian ODEs in spaces of probability measures as studied by Ambrosio and Gangbo. We also give a general formulation of the Stability Principle in a rigorous mathematical framework.

Hypoglycaemia in an adult male: a surprising finding in pursuit of insulinoma.

A 50-year-old man who had suffered several episodes of early morning sweats and tremors was diagnosed as having hyperinsulinaemic hypoglycaemia. Cross-sectional imaging and endoscopic ultrasound revealed no pancreatic lesion. A selective intra-arterial calcium infusion study showed a two-fold increase of insulin secretion after infusion of the splenic and superior mesenteric arteries. Laparotomy was performed, no lesion was identified after full mobilisation of the pancreas, and nothing was evident with intra-operative ultrasound. A distal pancreatectomy was performed. Microscopically, the pancreas exhibited diffuse islet cell hyperplasia consistent with nesidioblastosis. The patient remains euglycaemic eight months post-operatively.

Effects of norethandrolone on the transport and peripheral metabolism of thyroxine in patients lacking thyroxine-binding globulin. Observations on the physiological role of thyroxine-binding prealbumin.

Studies of the effect of norethandrolone on the transport and peripheral metabolism of thyroxine were carried out in four patients lacking thyroxine-binding globulin. Before norethandrolone administration, values for serum protein-bound iodine (PBI) were decreased (1.8 +/-0.5 mug/100 ml) and the proportion of free thyroxine increased (0.036 +/-0.008%). As a result, values for the absolute concentration of free thyroxine iodine were at the lower end of the normal range (0.63 +/-0.12 mmug/100 ml). During the control thyroxine-turnover study, the thyroxine distribution space was strikingly increased (18.2 +/-7.9 liters) and the fractional rate of thyroxine turnover moderately increased (17.1 +/-11.3%/day), as compared to the expected mean values for normal subjects. Therefore, calculated values for the daily rate of thyroxine clearance were increased even more, ranging between 255 and 500% of normal values. However, owing to the low PBI in these patients, the daily disposal of thyroxine iodine was similar to that expected in normals on the basis of age and weight. During the administration of norethandrolone, the thyroxine-binding capacity of the thyroxine-binding prealbumin increased strikingly in all patients, values averaging 162% of those found during the control period. This increase was associated with a highly significant increase in PBI (133% of control values) and a small but significant decrease in the proportion of free thyroxine, resulting in no significant change in the absolute concentration of free thyroxine iodine. In all four patients, administration of norethandrolone was associated with a pronounced decrease in the thyroxine distribution space to values which averaged 69% of those found during the control period. Values for the fractional rate of thyroxine turnover increased slightly. As a result, thyroxine-clearance rate decreased in all patients. Owing to the reciprocal changes in clearance rate and PBI, no significant change in total daily thyroxine disposal was observed. The present studies reveal that when the thyroxine-binding prealbumin is increased in patients lacking thyroxine-binding globulin, several indices of peripheral thyroxine transport and metabolism are altered. However, these changes were small, even in the absence of thyroxine-binding globulin. It is suggested, therefore, that the effect of changes in thyroxine-binding prealbumin would be even smaller in individuals in whom thyroxine-binding globulin is present.

Distinct patterns of dystrophin organization in myocyte sarcolemma and transverse tubules of normal and diseased human myocardium.

BACKGROUND: Genetic mutations of dystrophin and associated glycoproteins underlie cell degeneration in several inherited cardiomyopathies, although the precise physiological role of these proteins remains under discussion. We studied the distribution of dystrophin in relation to the force-transducing vinculin-rich costameres in left ventricular cardiomyocytes from normal and failing human hearts to further elucidate the function of this protein complex. METHODS AND RESULTS: Single- and double-label immunoconfocal microscopy and parallel high-resolution immunogold fracture-label electron microscopy were used to localize dystrophin and vinculin in human left ventricular myocytes from normal (n=6) and failing hearts (idiopathic dilated cardiomyopathy, n=7, or ischemic heart disease, n=5). In control cardiomyocytes, dystrophin had a continuous distribution at the peripheral sarcolemma, with concentrated bands corresponding to the vinculin-rich costameres. Intracellular labeling extended along transverse (T) tubule membranes. Fracture-label confirmed this distribution, showing significantly greater label on plasma membrane fractures overlying I-bands (I-band 4.1+/-0.3 gold particles/micrometer A-band 3.3+/-0.2 gold particles/micrometer mean+/-SE, P=0.02). Hypertrophied myocytes from failing hearts showed maintenance of this surface distribution except in degenerating cells; there was a clear increase in intracellular dystrophin label reflecting T-tubule hypertrophy. CONCLUSIONS: Dystrophin partially colocalizes with costameric vinculin in normal and hypertrophied myocytes, a distribution lost in degenerating cells. This suggests a primarily mechanical role for dystrophin in maintenance of cell membrane integrity in normal and hypertrophied myocytes. The presence of dystrophin in the cardiac T-tubule membrane, in contrast to its known absence in skeletal muscle T-tubules, implies additional roles for dystrophin in membrane domain organization.

Postnatal ultrastructural changes in the cat myocardium: a morphometric study.

In order to understand more clearly the basis for our previously observed postnatal age related changes in the mechanical performance of the cat myocardium we have carried out a quantitative ultrastructural analysis of right ventricular papillary muscles obtained from adult cats (1.8 to 2.3 kg), infant cats (16 days of age), and neonatal kittens (less than 72 hours old). Volume fractions were calculated for myofibrils, sarcoplasm, mitochondria, lipid, sarcoplasmic reticulum (SR), golgi, and nuclei. The myofibril content of neonatal fibres was significantly less than that of the infant or adult groups (P less than 0.05 and 0.005 respectively). In addition the infant fibres contained a smaller volume of myofibrils than the adult ones (P less than 0.001). The mitochondrial content of the neonatal fibres was also significantly less than that of either the infants' (P less than 0.005) or adults' (P less than 0.001). These data provide an anatomical basis for the progressive age related increase in mechanical performance of the cat myocardium, in postnatal life.

The expression of dystrophin-associated glycoproteins during skeletal muscle degeneration and regeneration. An immunofluorescence study.

The distribution and expression of dystrophin and three of the dystrophin-associated glycoproteins (DAG), alpha-dystroglycan (156 kDa DAG), beta-dystroglycan (43 kDa DAG) and adhalin (50 kDa DAG) in rat skeletal muscle were studied during a controlled cycle of degeneration and regeneration induced by the injection of a snake venom. Cryosections of muscle at various stages of degeneration and regeneration were labeled using monoclonal antibodies to the three glycoproteins and examined at fixed time points after venom injection. Adhalin and alpha-dystroglycan remained present at the sarcolemma throughout the entire cycle of degeneration and regeneration. beta-Dystroglycan, on the other hand, was lost from the sarcolemma by 12 hours and reappeared 2 days after venom injection when new muscle fibers were being formed. Dystrophin was not lost from the sarcolemma until 24 hours after venom injection and did not reappear at the membrane until 4 days. It is suggested that dystrophin and the glycoprotein complex are synthesized separately, both temporally and spatially, and only become associated at the plasma membrane during the later stages of regeneration. The expression of beta-dystroglycan in the regenerating muscle fibers was first seen at sites of newly forming plasma membrane that were closely associated with the old basal lamina tube. The basal lamina may therefore have a regulatory or modulatory role in the expression of the DAG.

Urocortin, corticotropin releasing factor-2 receptors and energy balance.

Although there is considerable information regarding the role of brain CRF in energy balance, relatively little is known about the role of urocortin (UCN), which is an equally potent anorexic agent. Therefore, the effects of intracerebroventricular (icv) administration of UCN (0.01-1 nmol/day) on food intake and body weight were assessed over a period of 13 days and compared with data from CRF-infused counterparts. Although both peptides dose dependently reduced food intake and weight gain, the effects of CRF were much greater in magnitude than those of UCN, particularly on body weight. Pair-feeding studies suggested that, while the effects of CRF on body weight could not be completely explained by appetite suppression, the effects of UCN appeared to be due to its initial impact on food intake. CRF increased brown adipose fat pad and adrenal weights, whereas it reduced thymus and spleen weights. CRF also increased serum corticosterone, triglyceride, FFA, and cholesterol levels, whereas it reduced glucose. UCN did not produce any consistent changes in any of these indices of sympathetic nervous system activation. Concurrent administration of the CRF(2)-selective antagonist, antisauvagine-30 (ASV-30) (30 nmol/day) completely reversed or attenuated the effects of UCN and CRF (1 nmol/day) on food intake and body weight. ASV-30 did not significantly attenuate any of the above CRF-induced changes in tissue weights or serum chemistry. These data suggest that the central CRF(2) receptor may primarily mediate the anorexic, but not the metabolic effects of CRF.

Systemically and topically administered leptin both accelerate wound healing in diabetic ob/ob mice.

Leptin is a 16 kD protein that is produced by adipocytes and induces weight loss in both normal and genetically obese ob/ob mice. ob/ob mice are obese, have multiple metabolic abnormalities, and exhibit impaired wound healing. Exogenous administration of leptin to these animals induces weight loss and corrects their metabolic defects. Leptin's effect on wound repair, however, has not been studied. Systemic administration of leptin at doses ranging from 0.1 to 10 mg/kg/day induced a highly significant acceleration in wound repair in ob/ob mice (p<0.0001), but not in db/db mice, indicating that leptin's effects on wound repair were mediated through the leptin receptor. We then investigated the possibility that leptin was acting directly at the wound site by administering leptin topically, and found that topical leptin also induced a dose dependent acceleration in wound repair (p<0.0001). In addition, we found that all forms of leptin receptor, including the signal transducing long form, were present in skin by RNase protection assay, and that leptin receptor localized to subcutaneous vessels of wounded skin by in situ hybridization. Finally, we investigated the possibility that leptin stimulated angiogenesis in wounds by analyzing wound hemoglobin and wound vessel density. Neither systemic nor topical leptin induced any significant changes in either parameter, suggesting that leptin accelerates wound repair by a mechanism other than stimulation of angiogenesis. In summary, both systemic and topical leptin accelerate wound repair in diabetic ob/ob mice, possibly via the direct interaction of leptin with its receptors in wounded skin, but do not appear to significantly stimulate wound angiogenesis. Further studies to better elucidate the mechanisms of leptin's effects on wound repair are warranted.

A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse.

The central melanocortin system is critical for the long term regulation of energy homeostasis. Null mutations of the melanocortin-4 receptor (MC4-R) are associated with hyperphagia, obesity, and accelerated longitudinal growth in mice and humans. However, little is known about the function of another central melanocortin receptor, the MC3-R. To assess the role of the MC3-R in energy homeostasis, the majority of the mc3r coding sequence was deleted from the mouse genome. In contrast to the MC4-R knockout, which exhibits increased food intake, increased somatic growth, and defects in metabolism, mc3r-/- mice exhibit an exclusively metabolic syndrome. Homozygous null mc3r mice, while not significantly overweight, exhibit an approximately 50% to 60% increase in adipose mass. Mc3r-/- mice also exhibit an unusual increase in respiratory quotient when transferred onto high fat chow, suggesting a reduced ratio of fat/carbohydrate oxidation. Furthermore, male mc3r-/- mice also exhibit an approximately 50% reduction in locomotory behavior on the running wheel, suggesting reduced energy expenditure.

Calcium channel blocker drugs and diabetic control.

Calcium channel blockers are now widely used and there have been case reports of hyperglycemia with nifedipine. In a double-blind, randomized, crossover study of the effects of 4 weeks of therapy, each with two dihydropyridine calcium channel blocker drugs, nifedipine and nicardipine, glucose tolerance, plasma insulin levels, and hemoglobin A1 were assessed in 20 patients with non-insulin-dependent diabetes (mean age 59 years). There was no significant difference in glucose tolerance on active therapy (AUC: control, 548.3 +/- 24.8; nifedipine, 559.3 +/- 41.0; and nicardipine, 589.3 +/- 40.3). Similarly, despite producing significant hemodynamic effects, these drugs produced no significant effect on plasma insulin and hemoglobin A1 levels. Calcium channel blocker drugs may be useful alternatives to thiazide diuretics and beta-blockers in the treatment of ischemic heart disease and hypertension, especially in patients with diabetes.

Remodelling of optic nerve myelin sheaths and axons during metamorphosis in Xenopus laevis.

Whole mounts and transverse sections of Xenopus optic nerves were examined with the light and electron microscopes before, during, and after metamorphosis. In stage 52--58 tadpoles, almost all myelin sheaths were circular in transverse sections. Early in metamorphosis (stages 60--61) large redundant myelin loops surrounded many large axons in central regions of the nerve. The loops subsequently were broken down into ovoids and lamellar segments that remained mostly within oligodendrocytes. These myelin changes were not observed in the chiasm or next to the eye. They were not associated with significant axonal degeneration and were no longer apparent in optic nerves of young frogs. Xenopus optic nerves also became shorter during metamorphosis. We therefore suggest that myelin sheaths with redundant loops which degenerate and disappear are being remodelled as the nerve decreases in length.

Neurosecretion of arginine vasopressin by an olfactory neuroblastoma causing reversible syndrome of antidiuresis.

A 26-year-old woman with the syndrome of inappropriate antidiuresis demonstrated complete recovery following the resection of an olfactory neuroblastoma. Tissue arginine vasopressin levels by radioimmunoassay, immunohistochemical staining of the tissue arginine vasopressin, postoperative normalization of plasma arginine vasopressin levels, and the clinical resolution are evidence in support of a neurally derived tumor being the direct source of neurosecretion of arginine vasopressin rather than neurohypophyseal secretion or secretion from non-neural tissues, as reported to date in the etiology of the syndrome of inappropriate antidiuresis.

Immunogold labelling of dystrophin in human muscle, using an antibody to the last 17 amino acids of the C-terminus.

Immunolabelling with a 10 nm gold probe was used to localize dystrophin at the ultrastructural level in human skeletal muscle. The primary antibody was raised against a synthetic peptide containing the last 17 amino acids at the C-terminus of dystrophin. Using this antibody, labelling was almost entirely confined to a narrow band enclosing 40 nm either side of the plasma membrane and including the membrane itself. Histograms of the position of the gold probe relative to the plasma membrane showed modes lying over the membrane itself or the extracellular face of the membrane. One interpretation of these results is that the C-terminus of dystrophin is inserted in the plasma membrane alongside the glycoproteins with which it is tightly associated. Histograms of the distances between gold probes displayed modes at approximately 120 nm in both transverse and longitudinal sections suggesting that dystrophin forms a lattice-like network adjacent to the plasma membrane.

Y1 and Y2 receptor selective neuropeptide Y analogues: evidence for a Y1 receptor subclass.

Neuropeptide Y (NPY), a 36-residue polypeptide produced abundantly in both nervous and peripheral tissues, appears to play a significant role in the regulation of diverse biological processes, including feeding behavior and cardiovascular and psychotropic functions. The actions of NPY are mediated through effective binding to specific receptors of which two, designated Y1 and Y2, have been well characterized. A shortened cyclic analogue of NPY, des-AA10-17-cyclo-7/21[Cys7,21]NPY, was shown to retain high affinity for both human neuroblastoma SK-N-MC and SK-N-BE2 cell types (expressing Y1 and Y2 receptors, respectively). Increasing the size of the ring (des-AA10-17-cyclo-2/27[Cys2,27]NPY) in the present study produced a high-affinity analogue (Ki = 3.0 vs 0.3 nM for NPY) that bound exclusively to Y2 receptors. Using the feedback from structure-activity relationships, we also describe the optimization of specific substitutions and bridging arrangements leading to the production of other truncated, high-affinity Y1 selective analogues which bind, as does NPY itself, in the low-nanomolar range. Of greatest significance, des-AA10-17-cyclo-7/21[Cys7,21,Pro34]NPY (11) was found to possess agonistic properties with an affinity comparable to that of the native NPY molecule when tested for its ability to inhibit norepinephrine-stimulated cAMP release in SK-N-MC human neuroblastoma cells. Compound 11 also caused an increase in blood pressure in anesthetized rats. However, in two central nervous system models of Y1 receptor function, stimulation of feeding and anxiolytic activity, this analogue was inactive, which suggests the presence of a new subclass of receptors. In summary, the present results demonstrate that residues 10-17 of NPY are not directly involved in either Y1 or Y2 receptor recognition or activation. This suggests that the selectivity of NPY receptors is highly dependent on subtle conformational changes such as the substitution of residue 34 to a proline or the introduction of intramolecular constraints. Additionally, we have produced an analogue of NPY that selectively activates peripheral NPY Y1 receptors.

Co-localization of dystrophin and beta-dystroglycan demonstrated in en face view by double immunogold labeling of freeze-fractured skeletal muscle.

An absence of dystrophin causes Duchenne muscular dystrophy, but the precise mechanism underlying necrosis of the muscle cells is still unclear. Dystrophin and beta-dystroglycan are components of a complex of at least nine proteins, the dystrophin-glycoprotein complex (DGC), that links the membrane cytoskeleton to extracellular elements in skeletal and cardiac muscle. Biochemical studies indicate that dystrophin is bound to other components of the DGC via beta-dystroglycan, which suggests that the distribution of these two proteins should be almost identical. In this study, therefore, we examined the spatial relationship between dystrophin and beta-dystroglycan with a range of different imaging techniques to investigate the extent of the predicted co-localization. We used (a) double immunogold fracture-label, a freeze-fracture cytochemical technique that allows high-resolution face-on views of labeled membrane components in thin sections and in platinum-carbon replicas, (b) double immunogold labeling of cryosections and (c) confocal microscopy. Both dystrophin and beta-dystroglycan were found over the entire fiber surface and, when labeled singly, the nearest neighbor spacing of labeling sites for the two proteins was indistinguishable. With double labeling, very close co-localization could be demonstrated. The results support the conclusion that dystrophin and beta-dystroglycan directly interact at the muscle plasma membrane. (J Histochem Cytochem 46:945-953, 1998)

A kinetic study of [131I] iodide and [99mTc] pertechnetate in thyroid carcinoma to explain a scan discrepancy: case report.

A study of unusual 1-131 and [99mTc] pertechnetate kinetics was carried out in a case of thyroid carcinoma. A major discrepancy in the handling of iodide, probably resulting from a deficient iodine organification process, was shown.

Plasma protein binding of lidocaine and warfarin in insulin-dependent and non-insulin-dependent diabetes mellitus.

We examined the plasma protein binding of an acidic drug (warfarin bound to albumin) and a basic drug [lidocaine (lignocaine) bound to alpha 1-acid glycoprotein] in 15 patients with insulin-dependent diabetes mellitus (IDDM) and 15 matched controls. We also examined protein binding of warfarin and lidocaine in 30 patients with non-insulin-dependent diabetes (NIDDM) and 25 controls. Compared with control, the binding of both warfarin (98.81 +/- 0.02 vs 98.57 +/- 0.03%, mean +/- SEM) and of lidocaine (69 +/- 2 vs 58 +/- 2%) was significantly reduced in IDDM. This group had lower concentrations of both albumin and alpha 1-acid glycoprotein (AAG), achieving statistical significance vs control for albumin only. In the patients with NIDDM, who had a similar level of glycosylated haemoglobin, while there was no significant difference in the binding of lidocaine there was a significant increase in warfarin binding compared with the control population (99.01 +/- 0.03 vs 98.82 +/- 0.04%). This study suggests that binding of both acidic and basic drugs is altered in both IDDM and NIDDM.

Plasma membrane cytoskeleton of muscle: a fine structural analysis.

The discovery of dystrophin and its definition as the causative molecule in Duchenne Muscular Dystrophy has led to a renewed interest in the molecular structure of the muscle fiber plasma membrane and its association with the extracellular basal lamina. The original identification of dystrophin gave credence to the possibility that the plasma membrane of the muscle fiber may be highly organized and involved in maintaining appropriate homeostasis in this actively contracting cellular system. In this review, we examine the currently known members of the muscle fiber plasma membrane cytoskeleton and the interactions that occur between the different members of this complex using histological, electron microscopic, and confocal methods. From our studies and others cited in this review, it is clear that the dystrophin cytoskeletal complex is not completely understood and component molecules continue to be discovered. Perhaps equally importantly, currently defined molecules (such as alpha-actinin or neuronal nitric oxide synthase) are being recognized as being specifically associated with the complex. What is striking from all of the studies, to date, is that while we are able to identify members of the dystrophin cytoskeletal complex and while we are able to associate mutations of individual molecules with disease(s), we are still unable to truly define the roles of each of the molecules in maintaining the normal physiology of the muscle fiber.

Freeze-fracture characterization of isolated myelin and axolemma membrane fractions.

The macromolecular organization of membranes isolated from the rabbit optic nerve and tract was analyzed using the freeze-fracture technique. A myelin fraction and two axolemma-enriched fractions were prepared from a preparation of myelinated axons isolated by flotation in a buffered salt-sucrose medium. In the myelinated axon preparation, axolemma and myelin membranes were easily identified. Larger areas of the axon membrane and myelin membrane totally lacked intramembranous particles. The particles remaining on the myelin membrane formed patches of evenly distributed elongated and globular particles. In contrast, the particles remaining on the axolemma were globular in shape and tightly clustered. Particle clustering and particle-free areas were not characteristic of either the axolemma or myelin membrane of whole nerves fixed in situ and processed for freeze-fracture. The isolated myelin membrane fraction contained a large number of vesicles completely lacking intramembranous particles. Of the remaining membrane vesicles, profiles with dispersed elongated and globular particles predominated. A small percentage of vesicles displayed intramembranous particles of the same size, shape and clustering pattern as that seen on the axolemma of the myelinated axon preparation. The two axolemma fractions were enriched in membrane containing tightly clustered globular particles. Particle-free vesicles as well as some myelin membrane vesicles were also seen in the axolemma fractions.