Biomedical subjects
Publications and source records attributed to G Löffler.
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A culture system for 3T3-L1 preadipocytes based on a serum-free chemically defined medium containing fetuin, transferrin, and pantothenate is described. In this system, adipose conversion depends on the following conditions. 1) In the presence of high insulin concentrations (1 microM), addition of corticosterone together with 1-methyl-3-isobutylxanthine (MIX) for not more than the first 4 days after confluence to the culture medium induces maximal adipose conversion within 12-14 days. MIX may be replaced by forskolin or permeable analogues of cAMP, indicating that its effect is due to elevated cellular cAMP levels. 2) At low insulin concentrations (1 nM), adipose conversion is reduced. Growth hormone or insulin-like growth factor I together with epidermal growth factor have to be present as a medium supplement together with corticosterone and MIX to get maximal adipose conversion. 3) The induction of adipose conversion by corticosterone and MIX in the presence of either high insulin concentrations or insulin-like growth factor I together with epidermal growth factor is accompanied by post-confluent mitoses. Inhibitors of DNA replication markedly reduce adipose conversion. Fibroblast growth factor and platelet-derived growth factor, although acting as potent mitogens on 3T3-L1 cells, do not support adipose conversion induced by corticosterone and MIX.
Obesity is regarded as a heterogeneous syndrome, which may appear in different forms. Various causes have been found to contribute to its pathogenesis. During recent years investigations of adipose tissue cellularity and its dynamic changes have gained growing interest. An important progress was the discovery of adipose tissue precursor cells. These cells have not yet been precisely identified by morphological and biochemical methods in intact tissue. However, due to methodological developments such precursor cells can be cultured both as primary cultures and as established cell lines. These culture systems have proven to be valuable models for the study of the processes involved in the formation of new fat cells.
Cell culture systems have proven to be valuable models for the study of the processes involved in the formation of new fat cells. Two separate steps may be distinguished in adipocyte development. First, the determination of a mesenchymal stem cell into a preadipocyte, second, its conversion into a mature fat cell. In cloned cell lines adipose conversion depends on at least one postconfluent mitosis possibly induced by insulin-like growth factors or by as yet unknown mitogens. In addition growth hormone, glucocorticoids, and insulin are needed for conversion to take place. The adipose conversion of preadipocytes originating from the stromal vascular fraction of adipose tissue does not depend on postconfluent mitoses and needs only insulin and glucocorticoid hormones in physiological concentrations. However, the ability to undergo adipose conversion is not stable in these cells, but gets lost after repeated subcultures or seeding at low densities. In addition to stimulating hormones an increasing number of factors inhibiting the conversion process have also been detected, the physiological function of which remains unclear at the moment.
The adipose conversion of 3T3-L1 fibroblasts depends on serum factors, one of which has been identified as GH. Human serum extracts obtained by heat treatment do not contain intact GH, as shown by RIA, but still support adipose conversion. After organic solvent extraction followed by reversed phase HPLC, two adipogenic fractions can be purified. Their identification as cortisol and cortisone follows from the observations that both glucocorticoid hormones are strongly adipogenic in physiological concentrations and elute upon reversed phase HPLC with retention times identical to those of the adipogenic fractions. After derivatization by acetylation, the adipogenic fractions purified from human serum again do not differ from C-21 monoacetyl cortisol and -cortisone with regard to adipogenic activity and behavior on reversed phase HPLC and TLC. Treatment of heat extracts from human serum with cortisol antibodies strongly reduced their adipogenic activity which could be restored only after addition of cortisol to the original concentration. We conclude from these results, that the adipogenic activity of human serum depends mainly on its glucocorticoid (as well as GH) concentration.
A reproducible cell culture system is described that allows the study of adipose conversion in fibroblast-like cells isolated by collagenase digestion of epididymal and perirenal adipose tissue from male rats weighing 70-200 g. Adipose conversion as measured by lipid accumulation and increase in glycerophosphate dehydrogenase (GPDH) activity during differentiation strongly depends on the density at which cells are inoculated and starts only when cells are confluent and when physiological amounts of corticosterone and insulin are added. beta-Estradiol, testosterone, thyroxine, triiodothyronine, and growth hormone do not affect the differentiation process. Methylisobutylxanthine added during the first 2 days after confluence, added with insulin and corticosterone, potentiates the effect of insulin on GPDH activity and accelerates triglyceride accumulation. The effect of methyl-isobutylxanthine seems to be mediated by increased cyclic AMP concentrations, inasmuch as it may be replaced by forskolin.
Adipose conversion of 3T3-L1 cells depends on adipogenic factors present in serum. In order to find their origin, adipogenic activity in extracts from adrenals, kidneys, testes, ovaries, liver, spleen, sceletal muscle and adipose tissue of the rat was studied. If tissues were homogenized with aqueous buffers at low pH, no adipogenic activity could be extracted. Treatment with chloroform/methanol followed by phase separation however revealed considerable adipogenic activity in the organic phase from adrenals, kidneys, ovaries, testes and sceletal muscle and additionally in the aqueous phase of liver. Further purification by liquid chromatography and reversed phase HPLC led to the identification of adipogenic activities from adrenals and kidneys as corticosterone and 11-dehydrocorticosterone. Adipogenic activity from liver in contrast is pronase-sensitive, exhibits an apparent molecular mass of 4 kDa and is probably a peptide.
The adipose conversion of 3T3-L1 fibroblasts to fully developed adipocytes depends on the presence of serum in the culture medium. This study demonstrates that human serum or plasma also effectively promote the adipose conversion of 3T3-L1 cells. At rising serum concentrations this effect is abolished however by the presence of antiadipogenic factors. Sera of newborns contained the highest adipogenic activity, approximately 1.8-fold higher than sera from lean adults. This difference is probably due to higher growth hormone (GH) concentrations. Adipogenic activity in sera from obese adults under reduction diet and in sera from older persons did not differ from that found in sera from lean young adults. Complete elimination of GH was achieved by heat treatment and ethanol precipitation. About 40-60 percent of the adipogenic activity in serum samples from adults and more than 70 percent of the activity in sera from newborns is due to GH. The remaining adipogenic activity was partially purified by gel chromatography at acidic pH. We conclude from these results that human serum contains at least two adipogenic factors: GH and a heat- and acid-stable low-molecular-weight component.
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The adipose conversion of 3T3-Li cells depends on a serum factor present in high amounts in fetal calf serum, which is heat stable and can be extracted from serum by ethanol precipitation. Sera of two genetically obese rodent species, fa/fa Zucker rats and C57Bl/KsJ-db/db mice, contain a high adipogenic activity which is very similar to that found in fetal calf serum. In contrast, sera of their lean siblings (Fa/Fa-Zucker rats and C57Bl/KsJ-+/+ mice) are devoid of adipogenic activity.
A murine monoclonal antibody (TU 110) prepared against blast cells of a patient with acute "undifferentiated" leukemia was tested in the microcytotoxicity assay on peripheral blood lymphocytes of 122 normal Caucasian donors. The TU 110 reactivity was found to show a correlation coefficient of 1.0 in population analysis for the presence of the HLA-B locus specificity B 13 as defined by alloantisera. Family segregation studies confirmed MHC linked inheritance of the TU 110 antigenic determinant strictly on HLA-B 13 positive haplotypes. As the first monoclonal reagent against the private specificity of this HLA-B locus antigen, TU 110 provides the possibility to study the structural relationships of sub- and supertypic determinants on this allotype and may help to correlate antigenic domains of HLA-B 13 with definable functional properties.
Control of quality and efficiency of surgical procedures performed is of utmost importance to every surgeon. This voluntary self-control has regularly been made by us for ten years. It allows accurate statements to be made on both the level of proficiency in surgery and the effectiveness of the surgical work done.
Isolated fat cells from rat brown adipose tissue in vitro respond to insulin with an increase of pyruvate dehydrogenase (EC 1.2.4.1) activity due to conversion of the inactive form of the enzyme (PDHb) to the active form (PDHa). Like in white adipocytes this effect depends on the presence of glucose or 2-deoxyglucose in the medium. The interrelationship between the steady state of the PDH-system and the phosphorylation state of the adenine nucleotides was studied in white adipose tissue. While insulin in the presence of 2-deoxyglucose caused a large fall of the tissue ATP/ADP ratio which could explain the increase of PDHa activity, the ATP/ADP ratio remained unchanged during incubations with insulin and glucose. Thus it appears that other factors than the ATP/ADP ratio are involved in the regulation of PDH activity by insulin the nature of which remains to be elucidated.
In isolated pancreatic acinar cells from the guinea pig stimulation of enzyme secretion by carbamoylcholine is slightly diminished in the absence of extracellular Ca. LaCl3 in a concentration, which does not influence the secretory response to carbamoylcholine, nearly completely abolishes 45Ca uptake by cells, indicating that Ca uptake is not necessary for secretion. In cells preloaded with 45CaCl2, addition of carbamoylcholine leads to an immediate release of 45Ca, which can be blocked by atropine or 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate and is not influences by LaCl3 in concentrations, which do not inhibit secretion. A similar release of 45CaCl2 from preloaded cells is obtained by addition of the mitochondrial inhibitors antimycin A, carbonylcyanide p trifluoromethoxyphenylhydrazone (FCCP), and oligomycin. Possibly due to markedly diminished ATP levels, neither antimycin A nor FCCP act as secretagogues, both compounds being inhibitors of secretion. Oligomycin, which decreases ATP levels only to 20%, stimulates secretion. Mitochondria and microsomes from pancreatic tissue are able to accumulate 45Ca. Mitochondrial 45Ca uptake can be driven by ATP or active respiration and is inhibited by NaN3, oligomycin, antimycin A or FCCP. Microsomal 45Ca uptake is ATP-dependent. NaN3 and mitochondrial inhibitors have no influence on microsomal 45Ca uptake, which is stimulated several-fold by oxalate. The results support the assumption, that in the guinea pig pancreas Ca mobilization from intracellular stores is necessary to initiate secretion. Due to their ability for an active accumulation of45Ca both mitochondria and microsomes could serve as intracellular calcium stores.
Evidence is presented for regulation by insulin of pyruvate dehydrogenase (pdh) interconversion in rat heart muscle in vivo and in vitro. In the alloxan diabetic rat the active (dephospho) enzyme amounted only to 12% of total PDH and was restored to 42% by insulin. Antilipolytic treatment of the dibetic animals was ineffective, indicating that the action of insulin was independent of a lowering of plasma non-esterified fatty acid concentration. On perfusion of isolated hearts from diabetic rats in the presence of glucose the proportion of pyruvate dehydrogenase in the active form remained low but was fully restored upon addition of insulin (2mU/ml) to the medium. No effect of insulin was obtained in the absence of glucose. The correlation between the rate of pyruvate decarboxylation in the perfused heart and of pyruvate dehydrogenase activity, in vitro, suggests that in the diabetic heart the entry of pyruvate into the citric acid cycle is largely controlled by covalent modification of the pyruvate dehydrogenase complex rather than by feedback inhibition. The possible role of insulin therein is discussed.
Plasma digoxin suspected to be elevated in anuric patients taking digitoxin was determined by radioimmunoassay in 15 anuric patients and 15 normal persons subjected to 0.1 mg digitoxin therapy per day. All plasma digoxin values from the anuric patients and the normal subjects were far below the lower limit of the therapeutic range of plasma digoxin. There existed no difference between the digoxin values determined in anuric patients and subjects with normal renal function; in both groups there was a scatter of digoxin values about the cross reaction line between digitoxin and digoxin antibody. It is concluded from the results that digoxin retention in anuric patients taking digitoxin plays an insignificant role; thus, the pharmacological effect is mediated by digitoxin itself.
Shifts in the distribution of the monovalent cations Na+ and K+ between the extra- and intracellular space seem to be important for the secretory response of the beta-cell. An attempt was therefore made to study the enzyme responsible for monovalent cation transport, the (NaK)-activated ATPase. In the presence of NaN3 as inhibitor of the mitochondrial Mg-ATPase, a NaK-ATPase with a specific activity of 72 mU X mg protein-1 could be demonstrated in crude membrane preparations of rat pancreatic islets. The enzyme, which was inactive in the absence of Mg++, needed both Na+ and K+ for activation and was inhibited by ouabain and PCMB. The main part of the NaK-ATPase was localized in the microsomal fraction. Glucose, sulphonylureas, somatostatin and diazoxide were without effect on NaK-ATPase.
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