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Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3-kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller-Harrison, R., Morin, M. and Czech, M. (1995) J. Biol. Chem. 270, 24442-24450). We report here that in response to insulin, activated complexes of IRS-1.PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1 adipocytes. The targeting of PI 3-kinase to rat adipocyte GLUT4-containing vesicles using vesicles prepared by sucrose velocity gradient ultracentrifugation was also demonstrated. Insulin treatment caused a 2.3-fold increase in immunoreactive p85 protein in these GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI 3-kinase activity in GLUT4-containing vesicle extracts increased to a similar extent. HPLC analysis of the GLUT4 vesicle-associated PI 3-kinase activity showed insulin-mediated increases in PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 when PI, PI 4-P, and PI 4,5-P2 were used as substrates. Our data demonstrate that insulin directs the association of PI 3-kinase with GLUT4-containing vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis that PI 3-kinase is involved in the insulin-regulated movement of GLUT4 to the plasma membrane.

In the frog, the fat body is the largest body lipid deposit and is associated with the gonad. The aim of the present work was to investigate the fine structure of the fat body at different periods of the annual cycle and during prolonged starvation. Results indicate that fat body cells of Rana esculenta caught in autumn and after winter hibernation resemble mammalian adipocytes of white adipose tissue and contain markers of adipose tissue, such as S-100 protein and lipoproteinlipase. However, unlike mammalian adipocytes, fat body adipocytes consistently show small lipid droplets associated with their single, large lipid deposits, a lack of a definite external lamina, and the presence of cellular prolongations and spicula at their surfaces. Transmission and scanning electron microscopy in association with lanthanum tracer experiments suggest that in fat body adipocytes a vesicular-tubular system connects the cytoplasm and the interstitial space. In June (i.e., during the reproductive period), fat body adipocytes appear to have lost much of their lipid deposit and adjacent adipocytes show interdigitation of their plasma membranes and prominent Golgi complexes. In starved frogs, fat body cells can be almost devoid of lipid and in regression to a near-mesenchymal state. Nevertheless, these fat bodies still contain lipoproteinlipase activity (approximately 45% of that found in lipid-filled ones), indicating persistent adipose differentiation of the cells therein. Results presented here show that the R. esculenta fat body is an adipose organ undergoing reversible extreme changes in adipocyte fat content, which are associated with definite ultrastructural features. The fat body represents a suitable model for studying adipose tissue under different and extreme physiological conditions.

Adipocyte lipid metabolism is primarily regulated by insulin and the catecholamines norepinephrine and epinephrine. Stimulation of the beta-adrenergic receptors (beta-AR) by catecholamines causes an increase in the rates of adipocyte lipid degradation and a decrease in the rates of lipid synthesis. These catabolic effects are in opposition to insulin, which causes net anabolic effects. Because most of the postnatal development of adipose tissue mass in pigs results from hypertrophy of adipocytes (rapid in first few weeks of life) caused by increased net synthesis of triacylglycerol, there is interest in the modulation of beta-AR in adipocytes of growing pigs. The beta-AR are characterized by measuring ligand binding to the receptor to ascertain the affinity of the ligand for the receptor and the receptor number. We found the affinity of the receptor did not vary with animal age (10, 28, and 75 d), with adipose tissue depot site, or in adipocytes of protein-deficient pigs. The beta-AR in obese pigs tended to have greater affinity than those in crossbred pigs of the same age and weight. The beta-AR number was not different when expressed per milligram of adipocyte membrane protein in pigs of different age, in obesity, in different adipose tissue depots, or during protein deficiency. The number expressed per cell or per unit adipocyte surface area did not differ between depots or during protein deficiency. The number per cell tended to be greater in the larger cells from 75-d-old pigs than in the smaller cells from 10- and 28-d pigs. It was greatest in obese pigs with the largest adipocytes. Under the various experimental conditions (age, obesity, depot, protein deficiency), the membrane fatty acid composition was greatly different, but in most cases there was no modulation of beta-AR affinity.

The basis of the chronic lipolytic effect of somatotropin on adipose tissue was investigated in sheep. Lipolytic rate was assessed in subcutaneous adipose tissue both in vivo, by microdialysis, and in vitro. Somatotropin treatment resulted in a small increase in basal (unstimulated) lipolysis and also in the maximum lipolytic rate observed in the presence of catecholamines both in vivo and in vitro. There was a small increase in the number of beta-adrenergic receptors but no change in the amount of the two isoforms of the stimulatory GTP-binding protein, Gs. Treatment with somatotropin decreased the response to antilipolytic agents such as the adenosine analog N6-phenylisopropyladenosine and prostaglandin E1. There was, however, no change in the number of adenosine receptors or amounts of the inhibitory GTP-binding proteins (Gi-1 plus Gi-2). Somatotropin also decreased prostaglandin E2 production by subcutaneous adipose tissue in vivo. Somatotropin treatment thus alters lipolytic regulation in sheep and this is characterized by changes in a number of proteins involved in this process.

Oxalyl thiolesters, a group of putative intracellular regulators, have been shown to be in vitro inhibitors of some cytosolic enzymes which are stimulated by insulin. In this study, the effects of insulin and oxalyl thiolesters on pyruvate dehydrogenase, beta-oxidation, and acyl-CoA hydrolase activities in mitochondria from rat epididymal adipocytes are compared. Using glutathione, CoASH, cysteine, and cysteamine as thiol sources, oxalyl thioesters were synthesized, purified, and quantitated. Mitochondria were isolated from rat epididymal adipocytes, some of which were incubated with or without insulin. Mitochondrial activities were determined by radioisotopic assay subsequent to control, insulin, or oxalyl thiolester incubation. Under the conditions used in this study, pyruvate dehydrogenase activity was increased 28% subsequent to 10-min incubation of adipocytes with 400 microU/ml insulin; in contrast, preincubation of adipocyte mitochondria with S-oxalylglutathione resulted in a dose-dependent 11-19% inhibition of pyruvate dehydrogenase. S-oxalylglutathione also attenuated the spermine-induced activation of pyruvate dehydrogenase. Insulin treatment resulted in a small but significant increase in beta-oxidation of palmitic acid while 100 microM S-oxalylglutathione mediated a 40% decrease in palmitate oxidation. Palmitoyl-CoA hydrolase activity was decreased 14% by insulin treatment; however, S-oxalylglutathione caused a 14-50% increase in hydrolase activity. The other oxalyl thiolesters were not as effective or as consistent as S-oxalylglutathione in modulation of the mitochondrial activities; free thiols and oxalic acid did not modulate the activities. In summary, pyruvate dehydrogenase, palmitate beta-oxidation, and palmitoyl-CoA hydrolase activities in adipocyte mitochondria were modulated in approximately equal but opposite directions by insulin and S-oxalylglutathione. These findings support the suggestion that oxalyl thiolesters may function as an intracellular signal recruited to return insulin to normal levels.

Two experiments were conducted to examine the effect of porcine growth hormone (pGH) on performance, carcase composition, muscle and fat deposition rates, muscle fibre characteristics, and fat cell volume in pigs. In the first experiment, ten pairs of littermates were treated with vehicle (saline buffer) or 80 micrograms pGH per kg live weight per day for 42 days starting at 50 kg live weight. In the second experiment, twelve pairs of littermates were untreated or treated with 3 mg pGH per day from approximately 56 kg live weight to slaughter at approximately 103 kg live weight. All pigs were fed ad libitum. In Experiments 1 and 2, respectively, feed intake decreased (10 and 11%) and the feed/grain ratio improved (8 and 13%), while daily gain was unaltered. There was an increase in deposition rates of muscle (11 and 22%), skin (27 and 23%), and bone (15% in both), and a decrease in deposition rates of intermuscular (48 and 24%) and subcutaneous (82 and 50%) fat. This resulted in a change in carcase composition towards more muscle (5 and 9%), bone (6 and 4%), and skin (18 and 12%), and less intermuscular (30 and 16%) and subcutaneous fat (51 and 32%). The increased muscle mass was due to enhanced hypertrophy of all muscle fibre types, while pGH did not affect the frequency of the different muscle fibre types. The reduction in subcutaneous fat was reflected in a similar reduction in fat cell volume. In contrast to the majority of pigs used in pGH experiments, the genotype used in the present experiments did not respond with respect to daily gain following pGH treatment. Furthermore, the increase in muscle deposition was rather low compared to results reported in pigs of other genotypes. These data together with published data on the cross-sectional area of muscle fibres indicate that genotypes with relatively large muscle fibres are less responsive to pGH treatment than genotypes with relatively small muscle fibres.

Vitamin A is stored in the lipid droplets of liver stellate cells (LSCs), as retinyl esters whose hydrolysis is necessary for the secretion of retinol into the blood. Here, we isolated these retinyl esters under their physiological form, i.e., in LSC lipid droplets, which had retained their morphological and biochemical characteristics. These retinyl esters are substrate for an hydrolytic enzyme, whose optimum pH is 4.1, and which is kinetically similar to the acidic retinyl ester hydrolase (aREH) we had previously described (Mercier et al., Biochim. Biophys. Acta (1994) 1212, 176-182). The cellular and subcellular localizations of aREH activity in rat liver suggest that this enzyme could be involved in the hydrolysis of the esterified vitamin A stores.

We report on eleven cases of a distinctive dermal spindle cell lipoma characterized by a mixture of mature adipocytes and spindle-shaped cells in a fibromucinous background. Six lesions showed a moderately well circumscribed plexiform pattern, five a well demarcated nodular pattern with compression of surrounding connective tissue and prominent stromal fibrosis. Clinically, the plexiform type mostly occurred in the thigh-groin-buttock area and the nodular type in the head-neck or acral location. While plexiform lesions were predominantly seen in middle-aged females, nodular types occurred in young adults of either sex. No recurrence was seen in five patients with follow-up. The tumour cells were vimentin positive and a thin cytomplasmic rim of S-100 protein positivity was seen in mature adipocytes. Ultrastructural studies revealed lipoblastic differentiation of spindle-shaped cells with lipid droplets and basal lamina formation. Dermal spindle cell lipomas seem to be the dermal counterpart of the most subcutaneously located spindle cell lipoma.

The expression of sodium-potassium pumps and glucose transporters in pure adipocyte plasma membranes from a hyperthyroid animal model was studied. Hyperthyroidism was induced by enteral administration of five doses of 90 micrograms of triiodothyronine every second day to 8-week-old rats. Following isolation of epididymal adipocytes, 3-O-methylglucose transport was measured and the number of Na/K-ATPase-(alpha 1- and alpha 2-isoforms) and glucose transporter (GLUT1 and GLUT4) molecules in sheets of adipocyte plasma membrane were determined by quantitative immunoelectron microscopy, using gold labelling. Maximal in vitro insulin stimulation of adipocytes increased the glucose transport rate and the amount of GLUT4 in the plasma membrane 15-fold, whereas the amount of alpha 2 was unaffected. In adipocytes from hyperthyroid rats, mean adipocyte volume was decreased by 18% and the quantities of GLUT4 per unit area of plasma membrane (maximal insulin stimulation) and of alpha 2 were decreased by 19% and 15%, respectively. Thus, hypotrophia of fat tissue in the hyperthyroid state is associated with a decreased expression in the plasma membrane of the glucose transporter GLUT4 and the alpha 2-isoform of Na/K-ATPase.

Chondroid lipoma is a recently described variant of lipoma with unusual morphologic features. Although classified as a fatty neoplasm, its phenotype is uncertain because it has not been determined whether cartilage is a real component or only simulated by light microscopy and whether the adipocytes demonstrate white or brown fat differentiation, issues that can be resolved only by electron microscopy. We present two cases of chondroid lipoma that ultrastructurally showed abundant intracytoplasmic lipid and glycogen and numerous pinocytotic vesicles, characteristic of white adipocytes. These findings support the conclusion that these tumors are composed solely of fat without true cartilage differentiation.

In the present study, the subtype specificity and species selectivity of the nonpeptide BIBP 3226, as well as its in vitro antagonism of neuropeptide Y (NPY)-mediated second messengers have been investigated. Radiolabeled NPY is potently displaced by BIBP 3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenylmethyl]-D- arginine amide] on human Y1 receptor expressing Chinese hamster ovary-K1 cells (Ki = 0.47 +/- 0.07 nM). SK-N-MC human neuroblastoma cells (Ki = 5.1 +/- 0.5 nM) and the rat parietal cortex membranes (Ki = 6.8 +/- 0.7 nM). The interaction of BIBP 3226 with the Y1 receptor is stereoselective, because the (S)-enantiomer of the (R)-configured BIBP 3226 displays almost no affinity (Ki > 10,000 nM). In contrast, concentrations up to 10 microM BIBP 3226 do not displace [125I]NPY from the human Y2 receptor (neuroblastoma cell line SMS-KAN), the rabbit Y2 receptor (kidney) and the rat Y2 receptor (hippocampus). Functional antagonism could be shown for the human Y1 receptor: 0.1 microM BIBP 3226 antagonizes the NPY induced Ca++ mobilization (pKb = 7.5 +/- 0.17) as well as the NPY-mediated inhibition of cyclic AMP synthesis (pKb = 8.2 +/- 0.24) in SK-N-MC cells. In contrast, none of the formerly described putative antagonists PYX-2, [D-Trp32]NPY and benextramine could be characterized as high affinity Y1 receptor antagonists. The 18 amino acid NPY analog EXBP 68 Ile-Glu-Pro-Orn-Tyr-Arg-Leu-Arg-Tyr-NH2, cyclic (2,4'), (2',4')-diamide] displayed Y1-selective affinity with in vitro antagonistic properties (Ki = 0.33 +/- 0.04 nM and pKb = 8.4 +/- 0.07) in SK-N-MC cells. Therefore, BIBP 3226 is the first potent and subtype-selective nonpeptide Y1 receptor antagonist.

The effects of starvation on fat cells and blood capillaries of the first abdomino-inguinal mammary gland in mice were investigated by light and transmission electron microscopy. The body weight of starved mice abruptly decreased to approximately 70% of that of controls at 3 days of starvation and, thereafter, gradually decreased. In adipose tissues of mammary stroma, multilocular fat cells increased in number and clustered during starvation to a glandular appearance at 6 days. Collagen fibers increased in amount around mammary ducts and buds. By electron microscopy, multilocular fat cells possessed numerous mitochondria, small lipid droplets, and plasmalemmal vesicles, while endothelial cells of the blood capillaries showed numerous pinocytotic vesicles plus short marginal folds and microvillous processes. These observations prove that the number of pinocytotic vesicles in blood capillary endothelium is closely related with the increased amount of lipid of fat cells in the mammary gland during starvation.

Bioptic fragments of adipose white tissue taken from trochanterian area from children of 2-22 months old were ultrastructurally investigated. Children were of both sexes, 5 normal and 22 with clinical diagnosis of malnutrition. There were studied many interadipocyte spaces signalling out in cases with malnutrition modifications of different components, some of them related with the degree of malnutrition. There were noted: disorganisation and disappearance of basal membranes of capillaries and glycolema; modifications of endothelial cells with lesions of the capillary wall and free degraded red blood cells; disorganization of the ground substance in small areas or sometimes extended to all matrix of the space; collagen fibres reduced in number and size, and in two cases the presence of collagen fibrils with severe lesions, realeasing an electrondense material, fibrinoid-like; matrix infiltration, in some cases with lipids. In only one interadipocyte space a synaptic button was noted in contact with capillary. In malnutrition lesions of cellular elements of the white adipose tissue the following were observed: adipocytes, fibroblasts, fibrocytes, endothelial cells, mast cells--which in their turn are responsible for modifications of macromolecular structures of the extracellular matrix--glycosaminoglycans, proteoglycans, components of which biosyntheses are cell-dependent.

Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

To clarify the effect of glucocorticoid on glucose transporters (GLUT) in adipocytes and muscle, we examined the changes of GLUT4 in rat heart muscle, skeletal muscle and adipocytes during long-term administration of dexamethasone and the translocation of GLUT4. The levels of GLUT4 in the plasma membrane and the low-density microsome fraction were measured by Western blotting using anti-GLUT4 peptide antibody. The levels of GLUT4 in the heart and skeletal muscles of rat were unchanged by treatment of dexamethasone. In the adipocytes the level of GLUT4 in plasma membrane was changed, but it was decreased in the low-density microsome fraction. Although adipocytes are less involved in blood sugar regulation than skeletal muscle, this finding suggests that glucose metabolism in Cushing's syndrome is affected partly by a decrease of GLUT4 in the adipocytes.

Immunocytochemistry was used to determine the intracellular location of perilipins in adipocytes and the occurrence of these proteins in tissues involved in triacylglycerol metabolism. Confocal microscopy and 3-dimensional analysis of 3T3-L1 adipocytes showed that perilipin immunofluorescence, present on the surfaces of all sized lipid droplets, appeared unevenly dispersed on the surfaces of many large lipid droplets. Electron microscopy revealed that immunogold staining for perilipin was located directly on the surface layer apposed to and surrounding the core triacylglycerol of intracellular lipid droplets of adipocytes in culture or from white and brown adipose tissue. Freeze-fracture electron microscopy indicated that the hydrophobic face of this surface monolayer contained particles identical in size and distribution to intramembranous particles (IMPs), which are unique structural features of the hydrophobic faces of bilayered membranes. Also, freeze-fracture replicas revealed areas of continuity between the surface layer of lipid droplets and the membrane leaflets of endoplasmic reticulum, suggesting that the droplet monolayer surface is an area of endoplasmic reticulum membrane leaflet modified by its unique content of perilipin. Microperoxisomes, identified by immunostaining for catalase, were found closely associated with lipid droplets, but external to and not in contact with the lipid droplet surface layer. Vimentin, identified by immunofluorescence, was present around the periphery of most lipid droplets in 3T3-L1 cells during early stages of adipocyte development but, in contrast to perilipins, vimentin was not around the periphery of many large lipid droplets in mature cells. Although perilipin was at the surface of lipid droplets in adipocytes of lactating mammary gland, none was found to be associated with the milk lipid droplets in alveolar epithelial cells, nor was the protein found on the surfaces of lipid droplets in hepatocytes. Studies in mammary gland show that perilipin immunostaining will be a valuable tool for the identification of tissue adipocytes severely depleted of their triacylglycerol stores and thus without their characteristic spherical shape. Perilipin's singular location on the surface monolayer of intracellular lipid droplets supports an intimate role for the protein in the triacylglycerol metabolic functions of adipocytes.

The cells which related to deposition of extracellular matrix (ECM) in human liver fibrogenesis were investigated with immunohistochemistry (IHC) and nucleic acid hybridization. Results showed that ECMs were high in chronic liver disease (CLD) tissues, particularly collagen III and its mRNA in active-CLD. In this group, cells positive for pre-collagen III pro-peptide (PIIIP) and collagen III mRNA were markedly increased and mostly located in the area connecting interstitium and parenchyma. These collagen producing cells were mainly the interstitial cells identified by IHC and electron microscopy as fat storing cells and associated myofibroblasts and fibroblasts. Their degree of hyperplasia was closely related to the infiltration of inflammatory cells.

Objective: The aim of this work was to study the reactivity to chronic cold stress of interscapular brown adipose tissue (IBAT) in old rats by stereological, immunohistochemical and ultrastructural methods., Design: Five 2-year-old rats were cold-acclimated at 4 degrees C for 4 weeks and six rats were used as controls (20-23 degrees C)., Measurements: The following were measured: IBAT volume and weight; unilocular and multilocular brown adipocyte content; preadipocyte number; multilocular cell mitochondrial area; cristae length and density per mitochondrion, and immunoreactivity for the brown adipose tissue specific uncoupling protein (UCP)., Results: Following cold acclimation, IBAT increased significantly in weight, volume, relative mass and number of multilocular adipocytes (170%). The number of unilocular adipocytes did not vary significantly. Multilocular adipocytes of both cold-acclimated rats and controls expressed the uncoupling protein, but in the experimental group cristae length and density per mitochondrion were significantly higher. Multilocular adipocyte precursors were observed in only one cold-acclimated rat but not in controls., Conclusion: The response of brown adipose tissue of old rats to chronic cold stimulus is similar to that observed in young and adult rats. Cold acclimation induces brown adipocyte recruitment: their number increases significantly, they test UCP-positive and their mitochondria are significantly more active than in controls. On the other hand, the number of unilocular adipocytes is not significantly affected, which may serve to improve the utilization of the heat produced by thermogenesis.

The relationship between glycosylation, dimerization, and heparin affinity of lipoprotein lipase (LPL) was studied in 3T3-L1 adipocytes. Three forms of LPL subunits were found in normal cells; totally endo H-resistant (57 kDa), partially sensitive (54 kDa), and totally sensitive (51 kDa) forms. LPL in normal cells was active, dimeric, and showed high affinity for heparin. LPL in cells treated with tunicamycin, preventing the transfer of N-linked oligosaccharide chain, was unglycosylated (51 kDa) and inactive. LPL proteins were found as an aggregate, and had low affinity for heparin. After treatment with castanospermine, an inhibitor of ER glucosidase I, 80% of LPL activity was inhibited. Most of LPL proteins were totally endo H-sensitive, present as an aggregate, and had low affinity for heparin. LPL in cells treated with deoxymannojirimycin, an inhibitor of Golgi mannosidase I, was active, dimeric, and had high affinity for heparin as in normal cells. But LPL subunits were all endo H-sensitive. These results suggest that core glycosylation and subsequent removal of glucose residue is required, but processing after Golgi mannosidase I is not necessary for dimerization and acquisition of high heparin affinity of LPL.

Involutional lipoatrophy is an apparent idiopathic lipoatrophy with characteristic histopathologic features. We report a patient with a distant history of intramuscular injections and subsequent typical involutional lipoatrophy in whom macrophage invasion of the fat was prominent. Light microscopy revealed small, thin lobules of fat with focally prominent blood vessels and a variably hyaline background. Macrophages containing granular acid mucopolysaccharide material were present in direct apposition to lipocytes, around involuting lobules and between collagen fibers in the neighboring dermis. Focal deposits of iron were observed. Electron-microscopic examination revealed macrophages abutting lipocytes and containing lipid droplets, clear vacuoles and lysosomes in varying proportions. Lipocytes varied in size. The lipid appeared normal in most, but scattered cells contained electron-dense granules or needle-shaped clefts within the lipid. We speculate that previous injections in our patient stimulated a macrophage response, with subsequent regression of lipocytes of the neighboring fat lobules.

The origin of brown adipocyte precursor cells is to date unknown. Some authors believe they arise from vascular cells, others from interstitial cells. The purpose of the present ultrastructural study was to find markers in rat fetal and perinatal adipose tissue that can be used to identify brown adipose precursor cells. The study was carried out on the interscapular brown adipose tissue of fetal (fetuses of 19 and 21 days) and perinatal rats (pups of 4 and 12 hours and of 1, 3, 5, 7, 9, 11, 13, and 15 days). The analysis focused on stem cells and showed the characteristic presence of typical mitochondria which make their identification as brown adipocyte precursor cells inequivocal. These cells were frequently observed in a pericytic position. Also some endothelial cells were characterised by typical mitochondria and abundant glycogen. These data seem to support the hypothesis that brown adipocytes originate from vascular cells.

We examined effect of insulin or 12-O-tetradecanoyl phorbol 13-acetate (TPA) on the subcellular redistribution of protein kinase C isoforms in rat adipocytes. Total Mono Q column-elutable novel PKCs (nPKCs) which are Ca(2+)-independent and phospholipid-dependent protein kinases, decreased in the cytosolic fraction and increased in the membrane fraction during treatment with insulin or phorbol ester for 10 min. Immunoblot analysis of novel PKCs, -epsilon, -delta and -zeta, showed that insulin stimulated the translocation of these PKC isoforms from cytosol to membrane, similar to the translocation of conventional Ca2+/phospholipid-dependent PKCs (cPKCs), -alpha, -beta, and -gamma. Phorbol esters stimulated the translocation of PKC-alpha, -beta, -gamma, -epsilon and -delta, but not PKC-zeta. These results suggest that (a) insulin and phorbol esters similarly stimulate the translocation of each PKC isoform except for PKC-zeta, and (b) the translocation of both nPKCs and cPKCs occurs during insulin and TPA actions in rat adipocytes.

Caveolae, also termed plasmalemmal vesicles, are small, flask-shaped, non-clathrin-coated invaginations of the plasma membrane. Caveolin is a principal component of the filaments that make up the striated coat of caveolae. Using caveolin as a marker protein for the organelle, we found that adipose tissue is the single most abundant source of caveolae identified thus far. Caveolin mRNA and protein are strongly induced during differentiation of 3T3-L1 fibroblasts to adipocytes; during adipogenesis there is also a dramatic increase in the complexity of the protein composition of caveolin-rich membrane domains. About 10-15% of the insulin-responsive glucose transporter GLUT4 is found in this caveolin-rich fraction, and immuno-isolated vesicles containing GLUT4 also contain caveolin. However, in non-stimulated adipocytes the majority of caveolin fractionates with the plasma membrane, while most GLUT4 associates with low-density microsomes. Upon addition of insulin to 3T3-L1 adipocytes, there is a significant increase in the amount of GLUT4 associated with caveolin-rich membrane domains, an increase in the amount of caveolin associated with the plasma membrane, and a decrease in the amount of caveolin associated with low-density microsomes. Caveolin does not undergo a change in phosphorylation upon stimulation of 3T3-L1 adipocytes with insulin. However, after treatment with insulin it is associated with a 32-kD phosphorylated protein. Caveolae thus may play an important role in the vesicular transport of GLUT4 to or from the plasma membrane. 3T3-L1 adipocytes offer an attractive system to study the function of caveolae in several cellular trafficking and signaling events.

Brown adipose tissue and collagenase-isolated brown adipocytes were investigated in rats by means of 1H and 13C nuclear magnetic resonance spectroscopy. After chloroform-methanol extraction of brown adipose tissue, proton and natural abundance 13C spectra of the chloroform fraction showed resonances attributable to triglycerides, and were qualitatively similar to those of the corresponding fraction of white adipose tissue. By means of quantitative analysis of 1H spectra, fatty acid unsaturation and polyunsaturation in triglycerides were found to be lower in brown than white adipose tissue; moreover, unsaturation parameters decreased in triglyceride fatty acids of brown adipose tissue upon norepinephrine administration or cold acclimatization of rats, and were affected by the age of donors. The molar percentage of mono- and polyunsaturated C18 fatty acids in triglycerides was determined from 13C spectra and found to change in the early post-natal period. Isolated, agarose-embedded brown adipocytes from 4-day-old rats showed a number of peaks in the carbohydrate region of 1H spectra that were not present in spectra of white adipocytes and almost disappeared in brown fat cells of older animals. These peaks could be restored by insulin exposure. Natural abundance 13C spectra of isolated brown adipocytes were resolved enough to allow unambiguous assignment of resonances to carbons of fatty acids, glycerol, glucose, ethanolamine, and choline. Calculation of the mono- to polyunsaturated fatty acids ratio in the cells was also performed. Nuclear magnetic resonance spectroscopy is a useful tool for the investigation of brown adipose tissue and adipocytes therefrom.

Rapid weight loss is frequently seen in malignancy. This weight loss is considered to result from enhanced lipolysis. Here, we show that adipocyte deletion by apoptosis, demonstrated by electron microscopy and DNA gel electrophoresis, occurs in some patients. Adipocyte apoptosis could not be demonstrated in patients without malignancy. These findings suggest that fat cell loss by apoptosis plays a role in malignancy-associated weight loss.

We have expressed the liver-type facilitative glucose transporter, GLUT2, in the insulin-sensitive 3T3-L1 adipocyte clonal cell line in an effort to address the importance of transporter isoform and cellular environment on the ability of insulin to mediate glucose-transporter translocation. Analysis of non-differentiated fibroblastic cell clones transfected with the GLUT2 cDNA identified the presence of this isoform in several independent clones. These clones exhibited increased deoxyglucose and fructose transport rates compared with control cells. Upon differentiation, the fibroblastic clones selected for study achieved > 95% phenotypic conversion into adipocytes. Expression of the GLUT2 protein was maintained throughout the differentiation protocol. Subcellular fractionation revealed that in response to insulin, unlike the native GLUT4, GLUT2 protein did not undergo significant translocation to the plasma membrane; furthermore, the subcellular distribution of the expressed GLUT2 was quite distinct from that of the endogenous GLUT4. 3T3-L1 adipocytes expressing GLUT2 only exhibited a 2-fold increase in insulin-stimulated fructose uptake, further suggesting that GLUT2 does not undergo insulin-stimulated translocation.

cAMP-binding ectoprotein (Gce1) and lipoprotein lipase (LPL) are anchored to plasma membranes of rat adipocytes by glycosylphosphatidylinositol (GPI) moieties as demonstrated by cleavage by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), reactivity with anti-crossreacting determinant antibodies (anti-CRD), and metabolic labeling with radiolabeled palmitic acid and myo-inositol. Quantitative release from the membrane of LPL and Gce1 requires both lipolytic removal of their GPI anchors and the presence of either 2 M NaCl or 1 mM inositol 1,2-cyclic monophosphate or inositol 1-monophosphate. PI-PLC-cleaved and released LPL or Gce1 reassociates with isolated plasma membranes of rat adipocytes and, less efficiently, with membranes of 3T3 fibroblasts. The specificity of the reassociation is demonstrated (i) by its inhibition after pretreatment of the membranes with trypsin, (ii) by its competition with inositol 1,2-cyclic monophosphate and inositol 1-monophosphate in a concentration-dependent manner, and (iii) by the limited number of binding sites. Enzymic or chemical removal as well as masking with anti-CRD antibodies of the terminal inositol (cyclic) monophosphate moiety of hydrophilic Gce1 and LPL significantly impairs the reassociation. These data suggest that in rat adipocytes GPI-proteins are not readily released from the cell surface upon lipolytic cleavage, but remain associated through a receptor which specifically recognizes the terminal inositol (cyclic) monophosphate epitope of the (G)PI-PLC-cleaved GPI moiety. This interaction may have implications for the regulated membrane release of GPI-proteins and for their possible internalization.

Brown adipose tissue and liver of hibernating, arousing and euthermic individuals of the dormouse Muscardinus avellanarius were studies using ultrastructural cytochemistry and immunocytochemistry with the aim to investigate possible fine structural modifications of the cell nucleus during the seasonal cycle. The general morphology of brown adipocyte and hepatocyte nuclei was similar in the three experimental groups. However, three nuclear structural constituents were identified only in hibernating individuals: coiled bodies (CBs) and amorphous bodies (ABs) were observed in hepatocytes and, together with bundles of nucleoplasmic fibrils (NF), were present in brown adipocytes of hibernating dormice. In arousing animals only some structural constituents suggestive of poorly structured CBs were found. The latter showed the same immunocytochemical features as CBs of hibernating individuals, suggesting that they are disappearing CBs. A possible involvement of CBs in storing and/or processing RNA which must be rapidly and abundantly released upon arousal is discussed. ABs similarly to CBs contain RNA and nucleoplasmic ribonucleoproteins (RNPs) and could also be involved in mRNA pathways. NF do not contain nucleic acids or RNPs and seem to be composed of protein-aceous material; their functional role in the nuclear metabolism of hibernating brown adipocytes remains unclear.

A novel modified chitosan carrying covalently linked imidazole groups (average molecular weight 700,000, degree of substitution 0.28, degree of acetylation 0.08) was used to stimulate bone formation in an animal model. Lesions (7 mm diameter) were surgically made in the femoral condyle of sheep and treated with the modified chitosan. Within 40 d after surgery, the neoformed tissue occluded the surgical hole and assumed a trabecular structure in the peripheral area of the lesion, while looking like a mineralization nodule in the central part in association with a fibrous component. In the control, no sign of osteoinduction or reparative process was observed and bone marrow was rich in adipocytes.

A 10-wk trial was conducted with 60 barrows and 60 gilts (initial weight = 47 kg) to investigate the effect of pST treatment (3 mg/d) for wk 0 to 5, 5 to 10, or 0 to 10 on performance, carcass traits, and adipose tissue cellularity of finishing hogs. The primary objective of the study was to determine the effect of 5 wk of pST treatment followed by 5 wk of withdrawal on performance and carcass traits relative to a control or to pigs treated for the entire period. After 5 wk, pST-treated pigs exhibited improved (P < .001) gain (11%) and efficiency (22%) and decreased (P < .001) feed intake (-22%) and 10th rib backfat accretion (-93%, P < .001). There was an increase in adipose cell number/gram of tissue (control, 2.57 vs pST, 3.36 x 10(6)/g; P < .005) and a decrease in cell diameter (control, 72.4 vs pST, 67.2 microns; P < .005). Backfat thickness and cell diameter increased in control pigs from wk 5 to 10. During the last 5-wk period, pST-treated animals continued to exhibit improved performance and maintained adipose cell size distribution and number similar to the pST-treated pigs at wk 5. Pigs treated for the first 5 wk and withdrawn from treatment for the second 5 wk had decreased efficiency (-12%, P < .05) and a twofold increase in backfat accretion (P < .001) relative to control. The increase in backfat thickness was accompanied by an increase in adipocyte diameter, indicative of lipid filling. Most of the benefits of pST observed during the first 5 wk were lost during the withdrawal period. Reductions in carcass fat in response to pST treatment are at least partially accounted for by inhibition of lipid filling in adipose tissue, an effect that is reversed upon cessation of treatment.

Large increases in fat stores involve an increase in adipocyte number via the replication and differentiation of preadipocytes, with the resultant cell gain widely regarded as irreversible. To date, there has been no clearly defined process or mechanism reported by which adipocyte deletion may occur. Here, we show that human adipocytes undergo apoptosis following growth factor deprivation or mild heat injury in vitro, thus demonstrating a cellular mechanism by which normal adipocyte loss could occur in vivo. The findings have implications for the understanding of adipose tissue kinetics and its derangement as well as for the potential development of methods for modifying fat store size.

The exposure of isolated adipocytes to insulin results in an approximately 20-fold increase in the rate of glucose transport into the cell. This increase is mediated by the movement of a pool of intracellular vesicles containing the so-called insulin-responsive glucose transporter (GLUT4) to the cell surface. In the resting state, most of the GLUT4 molecules are sequestered inside the adipocyte in an as yet unidentified intracellular compartment. TGN38 is an integral membrane protein which has been shown to be predominantly localized to the trans Golgi network [Luzio, Brake, Banting, Howell, Braghetta and Stanley (1990) Biochem. J. 270, 97-102]. Here we investigate whether GLUT4 and TGN38 are co-localized in the murine 3T3-L1 adipocyte cell line. Immuno-adsorption of intracellular vesicles containing GLUT4 with an anti-peptide antibody specific for this isoform did not deplete the low-density microsomal fraction of TGN38 in these cells; moreover, no TGN38 was detected in the GLUT4-containing vesicles by immunoblotting with a TGN38-specific antiserum. Immuno-adsorption of TGN38-containing vesicles and subsequent analysis of the proteins in these vesicles revealed that a detectable amount of GLUT4 (5-10%) did co-localise with TGN38. The amount of GLUT4 in the TGN38-containing vesicles did not change in response to insulin. Immunofluorescence analysis of TGN38 and GLUT4 in these cells revealed markedly different staining patterns. Reversal of insulin-stimulated glucose transport and subsequent analysis of the TGN38-containing vesicles demonstrated that during the re-cycling of GLUT4 to the intracellular storage site there was no increase in the amount of GLUT4 co-localized with TGN38. Taken together, these results suggest that the trans Golgi network is not the major site of the intracellular GLUT4 pool within 3T3-L1 adipocytes.

Purpose: Endothelial cell transplantation has been suggested as a method to improve the patency of prosthetic grafts used for vascular reconstruction. A major technical concern of all cell transplantation studies has been the purity of cells in the primary isolate used for subsequent transplantation. Accordingly we have evaluated the cellular constituents of liposuction-derived human fat with immunocytochemistry and scanning electron microscopy., Methods: Samples of liposuction-derived human fat were processed for immunohistochemistry and subsequently stained for the presence of von Willebrand factor (vWF), alpha-smooth muscle cell actin, cytokeratin (peptide 18), and the endothelial cell-specific marker EN4. We also performed histochemistry studies on the cells derived from this fat after collagenase dispersion of the liposuction far., Results: Immunohistochemistry revealed that 86.1% of the cells in intact, liposuction-derived fat express vWF, whereas 5.7% of the cells exhibited alpha-smooth muscle cell actin, and 1.0% expressed the mesothelial cell-related antigen, cytokeratin peptide 18. Expression of EN4 was found in 89.6% of the cells counted in intact far. After digestion of fat with collagenase and centrifugal separation of adipocytes from vascular and stromal cells, the expression of vWF, alpha-smooth muscle cell actin, and cytokeratin was 77.5%, 5.8%, and 2.1%, respectively. EN4 expression was observed in 74.6% of the isolated cells. Thus most cells present in liposuction-derived fat, even before tissue digestion and cell isolation, were characterized as endothelium. Although other cells common to mesodermally derived tissue were identified (e.g., adipocytes, smooth muscle cells, and mesothelium), they represented a minor fraction of the total cells present. On isolation, the number of cells expressing vWF- and EN4-specific antigens was less than that observed in intact fat., Conclusions: This finding suggests that a portion of cells reacting with antibodies in situ lose vWF and EN4 staining during the isolation procedure. Unlike omentum, liposuction-derived fat predominantly contains adipocytes and endothelial cells. On digestion of liposuction-derived fat and separation of cells, vascular endothelial cells represent the major cellular component.

Albumin is the major carrier of long chain free fatty acids, that are known to be heavily utilized by fat cells. To investigate the cellular structures involved in the interaction of albumin with preadipocytes, precursor fat cells isolated from rat epididymal fat pads were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and antibiotics. Cell differentiation was induced by 1.7 nM insulin added to the culture medium. The ensuing cells in various stages of differentiation were incubated with albumin, alone or bearing oleic acid, adsorbed to 5 nm gold particles (Alb-Au or Alb-OA-Au) for 10 min at 37 degrees C. As control, polyethyleneglycol-gold and IgG-gold complexes were used in same conditions. After extensive washing, the cell monolayer was fixed in 2.5% glutaraldehyde, and processed for electron microscopy. Examination of thin sections and morphometric analysis showed that: a) in the process of adipogenesis induced in vitro four cell types were simultaneously present: fibroblast-like cells (F), early adipocytes (EA), adipocytes (A) and aged adipocytes (AA); b) upon incubation with Alb-Au, the probe labelled preferentially coated pits and coated vesicles and only some uncoated vesicles; in time the tracer was found in endosomes, occasionally, multivesicular bodies and lysosome-like structures. The binding and uptake was dependent on the stage of cell differentiation, being less pronounced in fibroblast-like cells and increased in later stages (A and AA); c) Alb-OA-Au labelled the same structural features like the Alb-Au (i.e. coated pits and vesicles); however, the binding to the cell membrane was fourfold higher than that of Alb-Au in F and EA stages and was maintained at the same level in A and AA stages; d) control probes were taken up only by adipocytes and aged adipocytes, cells that express a strong phagocytic function for all tracers used. These results indicated that albumin bound to gold and especially albumin-bearing fatty acids-gold, bind preferentially to coated pits and vesicles of preadipocytes. Further internalization of albumin-gold particles seems to be non-specific, since both the probes and the controls adsorbed to gold were delivered to the same endosomal-lysosomal compartment.

Estrogen has various effects on adipose tissue. Although the presence of estrogen receptor (ER) has been demonstrated in rat adipose tissue and adipocytes, ER has not been identified in human adipose tissue. In this study, we demonstrated the existence of ER protein and ER messenger RNA (mRNA) in human sc adipose tissue and adipocytes. The cytosol fraction of human adipose tissue was partially purified by ammonium sulfate precipitation, and the presence of ER protein was analyzed by [3H]estradiol (E2) binding assay and Western blot analysis. [3H]E2 binding assay showed a low specific binding due to high nonspecific binding, and the dissociation constant (Kd) and maximal binding sites could not be obtained by Scatchard analysis. Western blots, however, showed the presence of ER protein in both the partially purified cytosol and nuclear fractions of human adipose tissue. The mol wt of ER in both fractions was approximately 66,000. Furthermore, Northern blot analysis of total RNA samples isolated from human adipose tissue showed the expression of ER mRNA at 6.2 kilobase in size. ER mRNA was also identified in isolated human adipocytes by the reverse transcription and polymerase chain reaction. These results indicated that both ER protein and ER mRNA are expressed in human adipocytes, suggesting that the effect of estrogen on human adipose tissues might involve a direct action.

The expression of the uncoupling protein (UCP), a protein unique to brown adipocyte mitochondria, was studied in sections of a human hibernoma by means of immunohistochemistry. Multilocular, but not unilocular, adipocytes expressed the UCP in the tissue. Further, the immunostaining was not uniform in multilocular cells, because small adipocytes with finely multivacuolar or scanty lipid deposit showed more intense staining. This pattern is similar to that found in brown adipose tissue. Ultrastructural investigation confirmed that a majority of proliferating cells had the morphological characteristics of brown adipocyte. Results indicate that adipocytes in hibernoma may be very close to brown adipocytes both morphologically and immunocytochemically.

The effects of alpha 2-adrenoceptor agonists (dexmedetomidine, oxymetazoline), alone or in combination with various alpha-adrenoceptor subtype-selective antagonists (CH-38083, idazoxan, WB4101, BRL44408, ARC-239, prazosin), on noradrenaline- and isoprenaline-induced lipolysis were investigated in human isolated abdominal subcutaneous fat cells. The rank order of potency of antagonists in preventing dexmedetomidine- and oxymetazoline-evoked suppression of isoprenaline-induced lipolysis was (pA2-values): CH-38083 (7.69 and 7.48) congruent to idazoxan (7.5 and 7.41) > BRL44408 (7.23 and 7.19) congruent to WB4101 (7.13 and 7.12) > prazosin (5.18 and 5.17) > ARC-239 (4.72, 4.9). While CH-38083 and idazoxan, non-subtype selective alpha 2-adrenoceptor antagonists and BRL44408, a selective alpha 2A-adrenoceptor antagonist as well as WB4101 potentiated the lipolytic effect of noradrenaline, ARC-239, the selective alpha 2B-adrenoceptor antagonist failed to affect it. In addition since the alpha 2A-adrenoceptor selective agonist, oxymetazoline concentration dependently inhibited the lipolytic effect of isoprenaline, and WB4101 and BRL44408 (alpha 2A-adrenoceptor antagonists) antagonised the effect of oxymetazoline in a competitive manner, it is concluded that the alpha 2A-adrenoceptor subtype is involved in antilipolysis. In addition, functional evidence was obtained that there is an interaction between alpha 2A- and beta-adrenoceptors located on the cell surface of adipocytes, through which locally released noradrenaline and/or circulating circulating adrenaline influence lipolysis.

1. A procedure is described for the separation of intact peroxisomes from human white adipocytes using a linear metrizamide gradient (20-50% w/v). 2. Peroxisomes were found in the high density region of the gradient in an intact form. 3. Mitochondria were distributed in the high density and low density regions of the gradient. 4. Lysosomes separated well from the peroxisomes, occurring only in the low density region of the gradient. 5. Low levels of glyoxylate cycle enzyme activities (isocitrate lyase and malate synthase) were detected within the light and heavy mitochondrial pellet fractions.

1. The existence of a functional beta 3-adrenoceptor in man was investigated by studying the lipolytic action of selective beta-adrenoceptor agents in isolated white omental and subcutaneous fat cells. 2. The non-selective beta 1/beta 2-adrenoceptor antagonist, CGP 12177 was lipolytic in both omental and subcutaneous fat cells. The intrinsic activity relative to isoprenaline was greater in omental than in subcutaneous cells. 3. Addition of the beta 2-adrenoceptor antagonist, ICI 118,551 and the beta 1-adrenoceptor antagonist CGP 20712A in combination or the non-selective beta-adrenoceptor antagonist propranolol alone (all 10(-7) M), induced a rightward shift of the dose-response curves for isoprenaline- and BRL37344-stimulated lipolysis of about 4 and 2 log-units, respectively. However, the antagonists did not alter lipolysis induced by CGP12177. 4. Several concentrations of beta-adrenoceptor antagonists were used to determine the pA2 values by Schild analysis. The values for CGP 20712A and ICI 118,551 (6.63 +/- 0.20 and 6.25 +/- 0.12) as antagonists of the lipolytic effects of CGP 12177 were over 2 units lower than the pA2 value for CGP 20712A against the response to the selective beta 1-agonist dobutamine (8.58 +/- 0.23) and the pA2 value for ICI 118,551 against the response to the selective beta 2-agonist terbutaline (9.15 +/- 0.26). 5. beta 3-Adrenoceptor mRNA expression, investigated with a polymerase chain reaction assay, was demonstrated in both types of adipocytes in the same cell preparations that had a lipolytic response to CGP 12177. 6. In conclusion, human white fat cells express an atypical beta-adrenoceptor in addition to beta 1- and beta 2-adrenoceptors. This receptor is stimulated more selectively by the beta1/beta2-antagonist CGP 12177 than by BRL 37344 and is poorly sensitive to blockade by selective beta1- and beta2-antagonists. On the basis of the pharmacological properties and the mRNA analyses, we suggest that this atypical receptor corresponds to the beta 3-adrenoceptor subtype.

To better understand the mechanism by which insulin exerts effects on events at the cell nucleus, we have studied insulin receptors and tyrosine kinase activity in nuclei isolated by sucrose density gradient centrifugation following insulin treatment of differentiated 3T3-F442A cells. Insulin stimulated nuclear accumulation of insulin receptors by approximately threefold at 5 min. The half-maximal effect was observed with 1-10 nM insulin. Following insulin treatment, phosphotyrosine content associated with the nuclear insulin receptor was also increased by twofold at 5 min with a similar insulin concentration dependency. These nuclear insulin receptors differ from the membrane-associated insulin receptors in that they were not efficiently solubilized with 1% Triton X-100. During the same period of time, insulin stimulated nuclear tyrosine kinase activity toward the exogenous substrate poly Glu4:Tyr1 tenfold in a time-dependent manner reaching a maximum at 30 min. The insulin receptor substrate protein 1 (IRS-1) could not be detected in the nucleus by immunoblotting. However, a nuclear protein with M(r) approximately 220 kDa was tyrosine phosphorylated, and insulin further stimulated this process threefold > 30 mins. Surface labeling was performed to determine if the nuclear insulin receptors would emerge from the plasma membrane fraction. Using 125I-BPA-insulin with intact cells, the intensity of nuclear insulin receptor labeling was negligible and not increased throughout 30 min incubation at 37 degrees C. In contrast, there was an increase in labeled receptors in the microsomal fraction following insulin treatment. Taken together, these results indicate that insulin rapidly increases nuclear insulin receptor appearance and activates nuclear tyrosine kinase activity. The insulin-induced accumulation of nuclear insulin receptors cannot be accounted for by internalization of surface membrane receptors. These effects of insulin may play an important role in action of the hormone at the nuclear level.

Hepatic lipocytes are perisinusoidal cells that have been thought to be analogous to tissue pericytes, a cell type with purported vasoregulatory properties. However, we and others have recently demonstrated that lipocytes acquire markers of smooth muscle cells or myofibroblasts only after liver injury, via a process termed "activation." In this study, we document lipocyte contractility on collagen lattices and examine the importance of activation in this process. In culture, lipocytes became contractile only after spreading and activating, coincident with expression of smooth muscle alpha actin, a marker of activation (1990. Virchows Arch. B Cell Pathol. 59:349). After 5 d in culture, lipocytes induced rapid and sustained contraction of collagen lattices (to 43.7 +/- 2.3% of their original size 24 h after detachment). There was no contraction of lattices containing hepatocytes. Scanning electron microscopy demonstrated intimate associations of lipocyte cell membranes and collagen fibrils. Reduction in cell volume during contraction was also prominent. Lattice contraction by lipocytes was proportional to cell number. Serum was a potent stimulator of lipocyte contraction, as were endothelin types 1, 2, and 3; the effect of serum and endothelin 1 were additive. Neither thrombin, angiotensin-II, serotonin, nor the cytokines PDGF and TGF beta induced contraction. Cytochalasin B treatment resulted in concentration-dependent inhibition of contraction. As a test of the in vivo relevance of the culture findings, lipocytes were isolated from fibrotic animals and examined immediately after adherence. Whereas lipocytes from normal liver were initially compact, smooth muscle alpha actin negative and noncontractile, cells from animals with hepatic injury due to CCl4 displayed an activated appearance, expressed smooth muscle alpha actin, and were contractile immediately after adherence. Additionally, IFN-gamma, an agent which blocks lipocyte activation (1992. Hepatology. 16:776), inhibited lipocyte contraction. The data document that normal (i.e., quiescent) lipocytes are not contractile, but that activation is associated with the development of contractility. These findings suggest that a role for lipocytes in organ contraction or vasoregulation may be confined to injured, not normal liver.

HGFu and Ob17 are cell lines derived from adipose tissue of lean (+/?) and ob/ob mice, respectively. Neither adenylyl cyclase activity nor G protein abundance and subcellular distribution have been assessed previously in these cells. Cyclase activity was low and resistant to catecholamine stimulation in both cell lines. However, the enzyme could be stimulated to high levels by forskolin and Mn2+. Gs alpha (largely the long isoform), Gi alpha 2, and G beta were the major G protein subunits identified. The levels of G protein mRNA expression were similar in both cell lines and, unlike actin expression, did not change as a result of differentiation. Immunoblotting and ADP-ribosylation of the G peptides corroborated these results. Assessment of the subcellular localization of the subunits by indirect epifluorescence and scanning confocal microscopy showed that each of the subunits had a characteristic subcellular pattern. Gs alpha showed vesicular cytoplasmic and nuclear staining; Gi alpha 2 colocalized with actin stress fibers and disruption of these structures altered the distribution of Gi alpha 2; beta subunits showed some colocalization with the stress fibers as well as a cytoplasmic vesicular and nuclear pattern. As a result of differentiation, there was reorganization of the actin, together with the Gi alpha 2 and beta fibrous patterns. Both cell lines showed similar modifications. The induction of differentiation in these cells is therefore not associated with changes in adenylyl cyclase activity nor of the abundance of G-protein subunits, although reorganization of some of these subunits does accompany actin reorganization.

Unlike hemopoietic cell, stromal cells of aplastic anemia patients' bone marrow undergo pathological shifts. This has been proved by ultrastructural investigations which showed intranuclear inclusions structurally similar to virus bodies. The latter are divided into three types this allowing follow-up of the inclusions to mature forms seen in the stromal cells cytoplasm. The findings suggest primary viral affection of stromal cellular elements of aplastic anemia patients' bone marrow. The attention is also drawn to ultrastructural shifts recorded in adipose cells in aplastic anemia.

The fine morphology, size, and perichromatin granule frequency were analysed in brown adipocyte nuclei from hibernating, arousing, and euthermic dormice, Muscardinus avellanarius. Unusual nuclear structural constituents such as nuclear amorphous bodies, coiled body-like constituents and bundles of nucleoplasmic filaments were described as typical of hibernating nuclei. Morphometrical findings showed significant difference in total nuclear and nucleolar size in the three physiological conditions investigated as well as decreasing frequency of perichromatin granules in nuclei of hibernating to arousing to euthermic animals. A possible involvement of these granules in the intranuclear transport or storage of pre-mRNA is discussed in the context of other experimental evidence.

We investigated the putative common nature of the rat atypical beta-adrenoceptors mediating white adipocyte lipolysis and proximal colon motility inhibition, using the non-selective antagonist alprenolol and agonist isoprenaline and the selective agonists SR 58611A and BRL 37344. Results in either isolated intestinal and fat tissues were consistent with: isoprenaline acting through both typical (beta 1, beta 2) and typical beta-adrenoceptors; SR 58611A and BRL 37344 acting solely through the latter. The identical pA2 values obtained with alprenolol, irrespective of the tissue and the selective agonist (SR 58611A or BRL 37344) used, support the high functional homology of the atypical beta-adrenoceptors in rat colon and adipocytes.

Two types of adipose cells were found in the connective tissue on day 7 after bone matrix gelatin (BMG) implantation and an injection of bupivacaine: mature adipose cells with a large lipid droplet (2-140 microns) and immature adipose cells with many small lipid droplets (0.1-2 microns). On day 10 after BMG implantation, typical adipose tissue was observed near the implant. The immature adipose cells had small, spherical mitochondria, glycogen granules and cytoplasmic microvesicles, and they might differentiate from undifferentiated mesenchymal cells in the connective tissue or the peripheral cells around the vessels as a white adipose tissue. These findings suggest that the differentiation of adipose cells in the connective tissue near heterotopic bone formation might be induced not only by mechanical and/or bupivacaine injury, but also by some factor or factors of the BMG.

A major glycoprotein fraction of the isolated adipocyte plasma membrane migrated in the 200,000-dalton region of dodecyl sulfate polyacrylamide gels following solubilization in 2% dodecyl sulfate/4M urea at room temperature in the absence of reductant. Limited heat treatment allowed resolution of the glycoprotein into two distinct bands in the same high-molecular-weight region plus a new 94,000-dalton glycoprotein band. Prolonged incubation of solubilized plasma membranes at 100 degrees C for 15-30 min or incubation with reductant resulted in complete conversion of the high-molecular-weight bands to the 94,000-dalton region, indicating dissociation of dimers to the monomeric form. When the dimer bands on column gels were electrophoresed in the second dimension on slab gels in the presence of reductant, no low-molecular-weight bands were observed other than that in the 94,000-dalton region. The effects of limited heat treatment to permit resolution of the two dimers and the extended treatment to convert the dimers to the monomeric form were markedly inhibited by alkylation of the solubilized membrane protein with N-ethylmaleimide or oxidation with H2O2 or diamide. However, these latter treatments did not prevent complete dissociation of dimers due to addition of reductants. These results suggest that two glycoprotein fractions may exist as dimers in the native fat cell plasma membrane. The data are consistent with a model in which the glycoprotein subunits are linked by hydrophobic bonds that are sensitive to reduction of intramolecular disulfides but are stabilized by alkylation or oxidation of the glycoprotein sulfhydryls.