Human Adipocyte Plasma Membranes Research Articles

Intérêt de l’analgésie locorégionale continue (ALRC) dans la réhabilitation du déficit neurologique douloureux. Expérience d’une coopération anesthésiste et centre de rééducation

Antisera were raised in rabbits against synthetic peptides corresponding to sequences of the guanine nucleotide binding proteins Gi1, Gi2, Gi3 and Go. These and previously described antisera were used to identify different G-proteins in Western blots of human adipocyte plasma membranes and to quantify them using purified recombinant α subunits as standards. Go was shown to be absent or ⪡ 15 pmol/mg of protein. A band stained by a previously characterized Go antiserum is suggested to be due to nonspecific staining of Gi1. Gi1 and Gi2 were the major G-proteins. Gi1 was present at concentrations of 52 and 18 pmol/mg of protein in lean and obese subjects, respectively, and the concentration was negatively correlated with the body mass index. Gi2 concentrations averaged 64 pmol/mg of protein and there was no correlation to the body mass index. Gi3 levels were much lower (⪡ 13 pmol/mg of protein) and the presence of this protein could not be demonstrated with certainty. The concentrations of Gi1 and Gi2 are thus over two orders of magnitude higher than those of the receptors whose effects they mediate. The low concentration of Gi1 in adipocyte plasma membranes of obese subjects could in part explain the attenuated inhibitory responses of adenylate cyclase in isolated fat cells in obesity.

Antibody-induced lysis of isolated rat epididymal adipocytes and complement activation in vivo.

To identify human monoclonal antibodies selectively binding to human adipocytes and to evaluate their ability to induce lysis of isolated rat adipocytes in vitro and to reduce rat complement levels in vivo. Using phage display technology, human monoclonal antibodies binding to human adipocyte plasma membranes were identified. Three antibodies (Fat 13, Fat 37, and Fat 41) were selected based on their additional cross-reaction with rat adipocytes and reformatted as a rat chimeric IgG2bs. The ability of these antibodies, both singly and in combination, to induce lysis of rat epididymal adipocytes in vitro and the reduction of serum complement levels in vivo in the rat was evaluated. All antibodies caused similar time- and dose-dependent lysis of isolated rat adipocytes. Calculated mean EC(50) values (maximum percentage of lysis in parentheses) were 0.680 microg/mL (63.2%), 0.546 microg/mL (72.4%), and 0.391 microg/mL (73.7%) for Fat 13, Fat 37, and Fat 41, respectively. Combinations were no more effective than individual antibodies in inducing lysis. Anti-adipocyte antibodies (both singly and in combination) were also similarly effective in vivo. In rats, doses of monoclonal antibody up to 10 mg/kg intraperitoneal generally caused almost complete depletion of serum complement up to 24 hours after dosing recovering to baseline values by day 5. Individual and combinations of monoclonal anti-adipocyte antibodies produced a complement-dependent and concentration-dependent activity to lyse adipocytes in vitro and in vivo as measured by a dramatic depletion in serum complement.

Inhibitory Effect of Isoproterenol on NADPH-dependent H2O2 Generation in Human Adipocyte Plasma Membranes Is Mediated by βγ-Subunits Derived from Gs

Previous studies revealed that human fat cell plasma membranes contain a multireceptor-linked H(2)O(2)-generating system that is under antagonistic control by hormones and cytokines and is stimulated by insulin via Galpha(i2). In this report, it is shown that the inhibitory action of the beta-adrenergic agonist isoproterenol is mediated by G protein betagamma-subunits, based on observations that its action was specifically reversed by anti-Gbeta antibodies or a C-terminal beta-adrenergic receptor kinase-1 fragment containing the Gbetagamma-binding site of the enzyme, and was mimicked by exogenously supplied G protein betagamma-subunits. Isoproterenol signals through a prototypical G(s)-coupled receptor. Consistent with these results, direct activation of G(s) by cholera toxin or by an anti-Galpha(s) antibody exhibiting beta-adrenergic receptor-mimetic properties (K-20) resulted in an isoproterenol-like inhibition of NADPH-dependent H(2)O(2) generation. In addition, a peptide corresponding to the target sequence of K-20 blocked the action of the catecholamine, apparently by competition between the peptide and G(s) for activated beta-adrenergic receptors, indicating that the G protein betagamma-subunits mediating the inhibitory effects of the catecholamine were in fact derived from G(s).

Insulin-induced Activation of NADPH-dependent H2O2 Generation in Human Adipocyte Plasma Membranes Is Mediated by Gαi2

Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.

β-Adrenergic responsiveness of adenylate cyclase in human adipocyte plasma membranes in obesity and after massive weight reduction

The aim of this study was to find out how β-adrenergic responsiveness of adipocytes is altered in obesity and by weight loss and to investigate what mechanisms lead to potential alterations in responsiveness. Crude plasma membranes were prepared from adipocytes of massively obese and normal-weight individuals, as well as previously obese patients that had lost an average of 38% of their initial weight after bariatric surgery. Stimulation of adenylate cyclase by isoproterenol varied considerably in fat cell plasma membranes from different individuals. Crude fat cell plasma membranes from obese patients were less responsive to isoproterenol than those from normal-weight subjects, whereas those from postgastroplasty patients were hyperresponsive. The response was correlated negatively with cell size and positively with β-adrenergic receptor density and with the ratio of β-receptors and stimulatory G-proteins (Gs). There was no correlation with Gs content. However, differences in receptor density between small and large cells or normal-weight, obese, and post-bypass patients could not explain the observed differences in responsiveness to isoproterenol between the different groups.

Quantitation of inhibitory G-proteins in fat cells of obese and normal-weight human subjects

Antisera were raised in rabbits against synthetic peptides corresponding to sequences of the guanine nucleotide binding proteins Gi1, Gi2, Gi3 and Go. These and previously described antisera were used to identify different G-proteins in Western blots of human adipocyte plasma membranes and to quantify them using purified recombinant α subunits as standards. Go was shown to be absent or ⪡ 15 pmol/mg of protein. A band stained by a previously characterized Go antiserum is suggested to be due to nonspecific staining of Gi1. Gi1 and Gi2 were the major G-proteins. Gi1 was present at concentrations of 52 and 18 pmol/mg of protein in lean and obese subjects, respectively, and the concentration was negatively correlated with the body mass index. Gi2 concentrations averaged 64 pmol/mg of protein and there was no correlation to the body mass index. Gi3 levels were much lower (⪡ 13 pmol/mg of protein) and the presence of this protein could not be demonstrated with certainty. The concentrations of Gi 1 and Gi 2 are thus over two orders of magnitude higher than those of the receptors whose effects they mediate. The low concentration of Gi1 in adipocyte plasma membranes of obese subjects could in part explain the attenuated inhibitory responses of adenylate cyclase in isolated fat cells in obesity.

Lack of beta 3-adrenergic effect on lipolysis in human subcutaneous adipose tissue.

Human adipocytes are responsive to both alpha- (antilipolytic) and beta-(lipolytic) actions of catecholamine agonists. These effects are mediated by G proteins which interact with adenylcyclase beta 1- and beta 2-adrenoreceptor (AR) subtypes are identified in human adipose tissue. The existence of a third beta-adrenergic (beta 3) receptor has been recently proposed. The beta 3-AR has been reported to mediate catecholamine-induced cAMP accumulation and to be more responsive in this regard than either the beta 1- or the beta 2-AR. To examine the possibility that a beta 3-AR plays a significant role in the control of catecholamine-stimulated lipolysis in human adipose tissue, we used a sensitive in vitro measure of lipolysis. Abdominal and gluteal sc adipose tissue samples from 14 adults were incubated with isoproterenol, a nonspecific beta-AR agonist which activates adenylyl cyclase via all three beta-AR subtypes, in the presence of varying concentrations of a beta-AR antagonist, alprenolol, which blocks the beta 1- and beta 2-AR, but not the beta 3-AR. In tissue from both abdominal and gluteal sites, alprenolol (10(-3) M) completely abolished the lipolytic response to isoproterenol (10(-6) M). Alprenolol (10(-3) M) alone reduced basal lipolysis by 10.2 +/- 2.2% (SEM) in abdominal tissue (P < 0.01), and 7.1 +/- 2.7% in gluteal tissue (P < 0.05). In the presence of alprenolol (10(-3) M), isoproterenol (10(-6) M) had no demonstrable lipolytic effect. The probability of incorrect acceptance of the null hypothesis that isoproterenol had no effect on lipolysis in alprenolol-blocked tissue is P beta < 0.05 for both depots. As an additional control, pooled inguinal adipose tissue from adult male Sprague-Dawley rats was incubated with the same concentrations of alprenolol. Rat adipose tissue has been previously shown to express the beta 3-adrenoreceptor and lipolysis was increased in rat adipose tissue in the presence of alprenolol 10(-4) M and 10(-5) M. We conclude that beta 3-ARs, if present in human adipocyte plasma membranes, do not significantly affect rates of lipolysis.

Characterization of human adipocyte adenosine receptors

125I-Hydroxyphenylisopropyl adenosine ( 125I-HPIA) was used to characterize adenosine receptors in human adipocyte plasma membranes. Steady state binding was achieved after 6 h at 37°. Scatchard plots were linear, with a K D of approx. 2.5 nM, and B max of 360–1800 fmol/mg protein. (−)N 6-phenylisopropyl adenosine (PIA) was a more potent inhibitor of binding than N-ethyl carboxamido adenosine, and (+)PIA was more than 10-fold less potent than (−)PIA, consistent with A 1 adenosine receptor binding. Theophylline was a potent inhibitor of binding (IC 50 approx. 10 μM). Photoaffinity cross-linking studies demonstrated that the receptor is a single subunit, M r approx. 43 kDa. The findings demonstrate that the human adipocyte adenosine receptor is similar to the A 1 adenosine receptor of rat adipocytes, although its molecular weight is higher, and its affinity for HPIA is lower than that of the rat.

Guanine-nucleotide-binding proteins Gi and Gs in fat-cells from normal, hypothyroid and obese human subjects

In human adipocyte plasma membranes, pertussis toxin catalysed the ADP-ribosylation of an apparently single 40 kDa protein. The same protein was also observed in Western blots by using an antibody which identifies the C-terminal decapeptide of Gi alpha (alpha subunit of Gi). In analogous experiments, cholera toxin and an antibody raised against the C-terminal decapeptide of Gs alpha (alpha subunit of Gs) were used to identify two proteins of 42 and 45 kDa, the former of which was more prominent. A method was developed to estimate the relative amounts of Gi and Gs in crude adipocyte plasma membranes in a single immunoblot by using the two antisera. In animal models, changes in the amounts of G-proteins have been suggested to explain alterations in hormone-responsiveness in hypothyroidism and obesity. However, the amounts of Gi and Gs were unaltered in thyroidectomized papillary-carcinoma patients who had been without hormone substitution for 4 weeks. In adipocyte plasma membranes prepared from markedly obese subjects, the amounts of both Gi alpha and Gs alpha as calculated per mg of protein were decreased, but the Gi/Gs ratio remained unaltered in comparison with control subjects.

Weight loss in massive obesity: Reciprocal changes in plasma HDL cholesterol and HDL binding to human adipocyte plasma membranes

Human obesity is frequently associated with elevated plasma triglyceride and cholesterol concentrations and reduced high density lipoprotein (HDL) cholesterol, abnormalities that commonly revert to normal levels with weight loss. This study was undertaken to examine possible mechanism(s) associated with the changes in plasma HDL cholesterol concentrations in massively obese patients after weight loss. Ten massively obese patients (two men and eight women, age = 37.8 ± 2.4 years) were studied before, during, and after 1 year of weight loss and weight maintenance following gastric stapling. Total cholesterol and low density lipoprotein cholesterol were within the normal range for sex and age before weight loss and did not change significantly during or after weight reduction. In the females, HDL cholesterol concentrations increased from 0.96 ± 0.06 mmol/L to 1.23 ± 0.3 mmol/L (mean ± SEM, n = 8, P < .05) with weight reduction. In the two men, plasma HDL cholesterol concentrations were, respectively, 1.22 and 0.65 mmol/L before and 1.23 and 0.98 mmol/L after weight loss. Specific binding of 125I-HDL 2 and 125I-HDL 3 to purified plasma membranes was determined using abdominal and omental fat depot before and after weight loss in six of the ten obese patients. An average reduction of 30% to 40% in 125I-HDL 2 and 125I-HDL 3 binding capacity to these membranes occurred after weight loss. Furthermore, a positive correlation ( r = .65, n = 10, P < .05) was observed between plasma HDL cholesterol and triglyceride concentrations before weight loss but not after weight loss ( r = .01). The changes in plasma HDL cholesterol and plasma triglyceride concentrations following weight loss were also not significantly related ( r = .55, n = 10). However the change in plasma HDL cholesterol correlated significantly with the change in HDL 2 binding to abdominal subcutaneous adipocyte plasma membranes ( r = 0.97, n = 5, P < .01). These results show that reduced plasma HDL cholesterol in obesity is not necessarily associated with hypertriglyceridemia. The increase in HDL cholesterol with weight loss and weight maintenance was gradual and delayed and is therefore not simply the result of acute nutritional adjustments. The reciprocal changes in HDL binding to adipocyte membranes suggest that fat tissue may participate directly in modifying plasma HDL cholesterol in obesity.

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL 3). This binding was independent of apolipoprotein E as HDL 3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL 3. To examine the importance of apolipoprotein A-I, the major HDL 3 apolipoprotein, in the specific binding of HDL 3 to human adipocytes, HDL 3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL 3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL 3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL 3, a 92 ± 3.1% reduction (mean ± S. E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL 3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [ 3H] cholesteryl ether labeled HDL 3, was also significantly reduced (about 35% reduction, P < 0.005) by substituting apolipoprotein A-II for A-I in HDL 3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.

Regional variation in high-density lipoprotein binding to human adipocyte plasma membranes of massively obese subjects.

Obesity is associated with significant changes in cholesterol and lipoprotein metabolism. High density lipoprotein (HDL) cholesterol is often reduced and adipose tissue cholesterol stores are increased in obese individuals. This prompted a study on the binding of HLD fractions (HDL2 and HDL3) to adipocyte plasma membranes obtained from massively obese subjects (BMI greater than 37 kg m-2) undergoing gastroplasty. Regional variation in HDL binding to these adipocyte plasma membranes was demonstrated. Membranes derived from the abdominal subcutaneous depot exhibited similar binding affinity (Kd) but higher binding capacity (Bmax) for HDL2 and HDL3 than that from the omental depot. There was significant inter-individual variation in Bmax but the amount of HDL2 or HDL3 bound to the two depots of the same individual was positively correlated (HDL2, r = 0.66, P less than 0.05; HDL3, r = 0.88, P less than 0.01). While HDL2 binding showed a higher affinity (lower Kd) than HDL3, a significant positive correlation existed between HDL2 and HDL3 binding to the same adipocyte membranes (r = 0.89, P less than 0.01). A significant inverse correlation (P less than 0.05) was also observed between HDL2 and HDL3 binding to adipocyte membranes and plasma HDL-cholesterol concentration. These results suggest that adipose tissue is an important site of HDL metabolism and the subcutaneous fat depot may play a proportionally more significant role due to its higher HDL binding capacity. It is further suggested that increased HDL binding and metabolism by the expanded adipose tissue mass may contribute to reduced plasma HDL-cholesterol levels frequently associated with obesity.

Enhanced binding of low and high density lipoproteins to human adipocyte plasma membranes: effects of temperature and proteases

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 degrees C (p less than 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner.

HDL binding to human adipocyte plasma membranes: regional variation in omental and subcutaneous depots.

Human adipose tissue contains one of the largest pools of exchangeable cholesterol in the body, accounting for about 25 percent of total body cholesterol in normal man (1,2). This adipose cholesterol pool is much expanded in obesity and in the massively obese state, well over half of total body cholesterol can be found in the fat tissue (1,2). Obesity is also commonly associated with a number of lipoprotein abnormalities. Increased synthesis and/or turnover of LDL (3) and VLDL (4,5) have been reported. In addition, HDL-cholesterol in plasma is significantly reduced (6–13) and has been attributed primarily to a decrease in the HDL2 subfraction (7).

Characterization of high density lipoprotein binding to human adipocyte plasma membranes

Characterization of high density lipoprotein binding to human adipocyte plasma membranes

Characterization of high density lipoprotein binding to human adipocyte plasma membranes.

Freshly isolated human adipocytes showed specific uptake of 125I-labeled human high density lipoprotein (HDL2 and HDL3), a portion of which could be released by subsequent incubation with excess unlabeled ligand. To study the mechanism of HDL binding, sucrose gradient-purified adipocyte plasma membranes were incubated with radioiodinated lipoprotein particles under equilibrium conditions in the absence (total binding) or presence (nonspecific binding) of 100-fold excess unlabeled ligand. Specific binding of HDL2 and HDL3, calculated by subtracting nonspecific from total binding, was Ca++ independent, unaffected by EDTA, and not abolished by pronase treatment of the membranes. Modification of HDL3 by reductive methylation or cyclohexanedione treatment also failed to affect its binding to adipocyte plasma membranes. High salt concentration (200 mM NaCl) inhibited specific binding of HDL2 and HDL3 but had no effect on LDL binding. A significant portion of 125I-HDL2 or 125I-HDL3 binding was consistently inhibited by adding excess unlabeled LDL, but this inhibition was incomplete as compared with a similar molar excess of unlabeled HDL2 or HDL3. The role of apoproteins (apo) in HDL binding to adipocyte membranes was examined by comparing binding of HDL2 and HDL3 isolated from normal, abetalipoproteinemic (abeta) and apo E-deficient (apo E0) plasma. Specific binding was observed with all normal and mutant HDL particles. Furthermore, a significant portion (61-78%) of abeta-HDL2, apo E0-HDL2, and apo E0-HDL3 binding was inhibited by adding 100-fold excess of unlabeled low density lipoproteins (LDL). The cross-competition of LDL and HDL binding was confirmed by the ability of normal, abeta, and apo E0-HDL2 to completely inhibit 125I-LDL binding. These data suggest that HDL binding is independent of apo E and that the responsible apoprotein(s) of HDL complete with LDL-apo B for binding to the same or closely related site in the adipocyte plasma membrane. Normal and apo E0-HDL3 binding was also completely inhibited by normal HDL2, which suggested that HDL2 and HDL3 probably bind to the same site. Scatchard analysis of normal HDL2, normal HDL3, and apo E0-HDL3 binding data best fitted a one-component binding profile with similar equilibrium dissociation constants (40-96 nM). HDL3 binding was found to be effectively inhibited by anti-human apo AI or anti-human apo AII, but not by anti-human apo B antisera. This binding was also unaffected by monoclonal anti-human apo B or E antibodies known to inhibit binding of apo B or apo E containing lipoprotein to the LDL receptor of cultured fibroblasts. These findings, taken together, suggest that human fat cells possess HDL binding sites with apo AI and /or apo AII specificity. The significant but partial inhibition of HDL2 and HDL3 binding by LDL along with the complete inhibition of LDL binding by HDL2 and HDL3 tends to exclude a single binding site that interacts both lipoproteins and favors the interpretation that LDL and HDL particles bind to multiple recognition sites or to different conformation of the same lipoprotein binding domain on the human fat cell.