Parathyroid hormone-induced lipolysis in human adipose tissue

Relative lipolytic activity of human parathyroid hormone-(1-34) (hPTH-(1-34], hPTH-(3-34), desamino-Ser1-hPTH-(1-34), and rat PTH-(1-34) was compared in human subcutaneous adipose tissues in vitro. Human PTH-(1-34), rat PTH-(1-34), and desamino-Ser1-hPTH-(1-34) stimulated in vitro lipolysis significantly above basal level at the concentration of 10(-6) M. Average increments of lipolytic rate were 2.39, 1.82, and 0.87 mumol/g per 2 hr, respectively, being significantly different among the three groups. On the other hand, hPTH-(3-34)-induced lipolytic rate was 0.83 +/- 0.18 mumol/g per 2 hr, not significantly different from the basal level (0.71 +/- 0.20 mumol/g per 2 hr). The effect of hPTH-(3-34) on glycerol release stimulated by hPTH-(1-34), isoproterenol, or forskolin was subsequently investigated. Human PTH-(3-34) produced a dose-dependent inhibition of hPTH-(1-34)-stimulated lipolysis. In contrast, isoproterenol- and forskolin-induced lipolytic rates were not influenced by hPTH-(3-34). The effect of propranolol on hPTH-(1-34)- or isoproterenol-induced lipolysis was also studied. Propranolol dose-dependently inhibited isoproterenol-induced lipolysis but had no effect on lipolysis stimulated by hPTH-(1-34). These results suggest that the amino acids at positions 1 (serine) and 2 (valine) of PTH are critical for the stimulation of lipolysis in human adipose tissue. Human PTH-(1-34) causes lipolysis after binding to receptors distinct from beta-adrenergic receptors of fat cells and possibly hPTH-(3-34) inhibits hPTH-(1-34)-stimulated lipolysis by competing at the level of PTH receptor.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.     

Ultrastructural studies on mitochondrial swelling

Mitochondrial swelling induced by valinomycin, calcium chloride and P(i) was studied after potassium permanganate fixation in suspension. Valinomycin induces a rapid K(+) influx, increase of the matrix space and out-folding of the cristae, with good preservation of the matrix material. This swelling is reversible but the cristae do not completely re-form and have a blebbed appearance. On repeated swelling and contraction cycles there is a gradual loss of matrix material. Calcium chloride and P(i) produce a slow swelling of the matrix space. Shrinkage induced by ATP was partial and not associated with return to the original structure.     

Relation of mitochondrial phospholipase A activity to mitochondrial swelling

Assays of mitochondrial phospholipase A activity and mitochondrial swelling demonstrated that the phospholipase A activity is related to the swelling under the experimental conditions used. Both were stimulated by added free fatty acid and CaCl(2), not affected greatly by the addition of monoacyl phosphoglycerides, and inhibited by EDTA. The amount of fatty acid hydrolyzed from endogenous phosphatidyl ethanolamine and phosphatidyl choline during swelling was calculated to be 20-30 times less than the amount of added free fatty acid that gave comparable swelling. Under the experimental conditions about 4% of the phospholipid was hydrolyzed. Mitochondrial swelling was studied by electron microscopy and turbidity measurements. The results found were in agreement, whether oleic acid was present or not, except for those values obtained after very brief incubation (1 min) and after incubation for longer than 35 min. The lack of direct proportion between swelling and the concentration of lysosomes present indicated that the swelling is related mainly to mitochondrial phospholipase A, although swelling due to contaminating lysosomes cannot be excluded entirely. The temperature dependence of spontaneous, fatty acid-induced, or CaCl(2)-induced swelling suggested that enzymatic activities are responsible for swelling.     

A mathematical model of mitochondrial swelling

Background

Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.

Results

Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2₁₉₇, nsP3₂₆₃ and nsP3₃₂₃ severely reduced infectivity, a serine to proline mutation in E2₂₀₆ appeared to enhance the virus titer production.

Conclusion

We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.     

The role of malate in hormone-induced enhancement of mitochondrial respiration

Shortly after the injection of glucagon, epinephrine, norepinephrine, vasopressin, or angiotensin II into fasted rats, mitochondria isolated from their livers contained elevated concentrations of malate and oxidized citrate, alpha-ketoglutarate, and, in some cases, succinate more rapidly than mitochondria from fasted, control rats. The administration of tryptophan, lactate, or ethanol and refeeding of rats fasted 24 h result in similar elevations of mitochondrial malate concentration and oxidation of added substrates. Treatments that resulted in elevated mitochondrial malate resulted also in increased uptake of added citrate, alpha-ketoglutarate, pyruvate, and, in some cases, succinate. It is postulated that the well-documented effect of gluconeogenic hormones on mitochondrial oxidation of carboxylic substrates may be mediated by malate which not only yields oxalacetate to support the tricarboxylic acid cycle but also facilitates the transport of added substrates, and which is regenerated in the tricarboxylic acid cycle.     

Top down analysis ceramide-induced mitochondrial dysfunctions: role of mitochondrial swelling

Mitochondrial role in ceramide-induced apoptosis pathway remains unclear. Direct effects of ceramide on mitochondria (cytochrome c release, respiratory chain inhibition, oxygen radicals production...) have been reported [1, 2] and we previously showed that addition of ceramide to intact cells or isolated mitochondria triggers mitochondrial swelling which appeared to be insensitive to cyclosporin A (CsA) [3, 4]. The purpose of this work was to determine to which extent this CsA-insensitive mitochondrial swelling, therefore distinct from permeability transition, participates to ceramide-induced apoptosis. To achieve this, we applied Top-Down analysis of integrated mitochondrial function [5], in order to better understand ceramide-induced mitochondrial dysfunctions.     

Parathyroid hormone-induced alterations of protein content and phosphorylation in enriched apical membranes of opossum kidney cells

Parathyroid hormone (PTH) reduces Na/Pi co-transport activity in opossum kidney (OK) cells in a process mediated by protein kinases A and C. Further, inactivation of Na/Pi transport involves irreversible inhibition, possibly via internalization, of the transport system. This study analyzed alterations of concentration and phosphorylation of membrane proteins of an apically enriched preparation induced by short (10 min) and long (3 h) term incubation with 10(-10) M PTH of monolayer cultures of the OK-cell line. To this end, an apically enriched membrane fraction was isolated from cells grown on Petri dishes and analyzed by two-dimensional gel electrophoresis. Long term exposure of the cells to PTH induced changes in apical protein concentration. Four proteins were found to be decreased and one protein was found to be increased in its concentration. Addition of 10(-10) M PTH to the cells led to transient phosphorylation of five proteins. In contrast to transient phosphorylation, phosphorylation of one protein increased over the time period of 3 h. Combined analysis of silver staining and autoradiography led to the detection of an acidic 35-kDa protein in which specific phosphorylation increased over a time period of hours. The results document for the first time alterations in apical membrane protein content and phosphorylation state mediated by PTH when added to an intact cellular system. It is concluded that the identified proteins represent possible candidates for being involved directly or indirectly in PTH alterations of membrane transport.     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.     

Understanding ion transport by quantification of mitochondrial swelling

Suspensions of mitochondria are turbid and scatter light. An increase in the matrix volume (swelling) due to the influx of permeable solutes results in a decrease in the amount of light scattered. This property can be used to study solute fluxes across the mitochondrial inner membrane. A rapid method for isolating mitochondria is presented along with three swelling experiments using energized and non-energized mitochondria to illustrate ion transport across energy transducing membranes.     

Studies of copper ion-induced mitochondrial swelling in vitro

1. A study of the mode and mechanism of Cu(2+)-induced mitochondrial swelling was carried out. 2. Mitochondrial swelling curves (E(520) turbidity changes) were obtained as a function of [Cu(2+)], pH, temperature and mitochondrial protein concentration. ED(50) was approx. 70mmumoles of Cu(2+). Calculation of the activation energy from the Arrhenius equation gave a value of 22900cal./mole per degree with Q(10) 4.02. 3. No lipid peroxides were formed during swelling. 4. Changes in oxygen consumption (Clark-type electrode) were dependent on the substrate used, but revealed no increased uptake in presence of Cu(2+). 5. Cu(2+)-induced swelling was inhibited by EDTA, 8-hydroxyquinoline, cyanide, citrate, bovine serum albumin, ATP, glutamate, GSH, dithiothreitol and sucrose. Azide, Amytal, antimycin A and oligomycin had no significant effect. Potentiation of swelling was seen with ascorbate, 2,4-dinitrophenol and succinate. 6. The occurrence of different types of mitochondrial swelling and the suggestion that Cu(2+)-induced swelling is mediated through a stoicheiometric interaction with a thiol-containing membrane receptor are discussed.