Hormone sensitive lipase expression and adipose tissue metabolism show gender difference in obese subjects after weight loss.

OBJECTIVE: The effect of weight reduction on hormone sensitive lipase (HSL) and lipoprotein lipase (LPL) gene expression and their relationship with adipose tissue metabolism were studied in massively obese men and women. SUBJECTS: Seventeen obese subjects (eight men, nine women) participated in the study (age 44+/-2 y, weight 145+/-8 kg, fat 40+/-2% of body mass, mean+/-s.e.m.), who were going through a gastric-banding operation for weight reduction. MEASUREMENTS: HSL and LPL mRNA expressions were analyzed using the reverse transcription competitive polymerase chain reaction. Subcutaneous fat lipolysis was measured in vivo by microdialysis and in vitro in isolated subcutaneous abdominal adipocytes. Measurements were done before and after 1 y of weight reduction. RESULTS: Significant reductions in weight (for men -20.3+/-2.5%, for women -18.3+/-2.1% (mean+/-s.e.m.) and fat mass (for men -27.6+/-7.9%, for women -21.8+/-3.9%) were observed in both genders. In women HSL mRNA expression decreased by 31% (P=0.008) and LPL expression increased slightly, but nonsignificantly (42%, P=0.110). These changes were not observed in men. In men, inhibition of lipolysis with alpha(2)-adrenergic and adenosine agonist was improved (P=0.001) in isolated adipocytes. CONCLUSIONS: This study uncovers new differences between genders in adipocyte metabolism along with weight reduction. In women, the observed changes in HSL and LPL gene expression suggest that deposition of lipids into adipose tissue might be favored after weight reduction. In men, the results indicate improved responsiveness to inhibition in adipose tissue metabolism along with weight reduction.

Concordance of in vivo microdialysis and in vitro techniques in the studies of adipose tissue metabolism.

BACKGROUND: Adipose tissue metabolism can be investigated directly in vivo by microdialysis and indirectly in vitro using isolated adipocytes. The in vitro studies are relatively easy to make and they give information about specific tissue metabolism. The in vivo studies, on the other hand, are supposed to give relevant data about tissue physiology interacting with other metabolic systems at the body level. OBJECTIVE: To investigate the concordance between the results on responsiveness to stimulation of lipolysis from in vivo microdialysis and in vitro isolated adipocytes. SUBJECTS: Altogether 22 massively obese otherwise healthy subjects (seven men and 15 women, age 41 (26-55) y, BMI 51.5 (37.5-73.9)kg/m2, mean (range)) going through the gastric banding operation participated in the study. METHODS: The microdialysis study was done after an overnight fast at rest. Lipolysis was stimulated with isoprenaline that was perfused into the subcutaneous abdominal adipose tissue. Local blood flow was estimated by ethanol dilution method. Adipose tissue biopsy for the in vitro study was taken from subcutaneous abdominal region during the operation. Lipolysis in freshly isolated adipocytes was stimulated with different concentrations of adrenaline or isoprenaline. RESULTS: Significant positive correlations were observed between the values of relative stimulation of lipolysis in isolated adipocytes and in the microdialysis study. These correlations improved after correcting for cell size or fat mass. CONCLUSION: The microdialysis study in vivo and lipolysis assay with isolated adipocytes in vitro provide concordant and complementary information of adipose tissue metabolism in the same individual.

Mechanisms of thyroid hormone control over sensitivity and maximal contractile responsiveness to beta-adrenergic agonists in atria.

This paper discusses the mechanisms of two basic effects of thyroid hormones on atrial responses to beta-adrenergic agonists, i.e. increased inotropic sensitivity and decreased maximal contractile responsiveness. The increased sensitivity of atria to beta-adrenergic agonists under thyroid hormones appears to be related to increases in beta-adrenoceptor density and Gs/Gi protein ratio, leading to activation of Gs-mediated pathway, but suppression of Gi-mediated pathway of adenylate cyclase regulation. Therefore, the i/c concentrations of cAMP and corresponding inotropic responses achieve their maximums at lower doses of beta-adrenergic agonist. Thyroid hormones also decrease the expression of phospholamban, but increase the expression of sarcoplasmic reticulum Ca2+-pump. As a result, the basal activity of sarcoplasmic reticulum Ca2+-pump increases, but its beta-adrenergic activation through phosphorylation of phospholamban decreases. It is suggested that these changes are causal for decreased maximal inotropic and lusitropic responses of atria to beta-adrenergic agonists.

Membrane-bound phosphodiesterases in rat myocardium.

Isoenzyme-specific phosphodiesterase (PDE) inhibitors are potential positive inotropic drugs. For evaluating such drugs in experimental models and to understand the physiological roles of the different isoenzymes, it is necessary to know what isoenzymes are present in the tissues studied. Rat myocardium has been reported to be devoid of the particulate cGMP-inhibited cAMP-PDE (type III isoenzyme). Here we re-evaluate the isoenzyme profile of rat myocardium. The cAMP-PDE isoenzyme patterns were studied by ion-exchange chromatography using siguazodan and rolipram, specific inhibitors of type III and IV isoenzymes, respectively. In contrast to earlier reports, type III isoenzyme was abundant in the particulate fraction. PDE III-specific antibodies depressed PDE activity and stained bands in Western blot with molecular masses 64 and 71 kDa. Type III isoenzyme of myocardial membranes was found to be unstable at 37 degrees C which may explain why earlier investigators have failed to demonstrate its presence. The data presented in this paper show that rat heart particulate fraction contains two low Km PDE isoenzymes, type III and type IV, in equal activities. Thus, in contrast to previous reports, this paper clearly shows the presence of considerable amounts of membrane-bound type III PDE isoenzyme in rat myocardium.

Alpha 2A-adrenergic regulation of cyclic AMP accumulation and lipolysis in human omental and subcutaneous adipocytes.

OBJECTIVE: To characterize differences in alpha 2-adrenergic regulation between subcutaneous and omental adipocytes which could offer a possibility of pharmacological intervention in the metabolic syndrome. DESIGN: Both subcutaneous and omental adipocytes were isolated from 32 patients. Adipocytes were incubated in the presence of adrenoceptor agonists, and cyclic AMP and glycerol levels were measured. alpha 2-Adrenoceptors of isolated plasma membranes were characterized. RESULTS: Adrenaline increased cyclic AMP levels about two-fold in omental adipocytes but had almost no effect in subcutaneous fat cells. The inhibition of cyclic AMP accumulation and glycerol release by UK-14304 and dexmedetomidine was less pronounced in omental adipocytes. The maximal effect of isoprenaline on cyclic AMP levels and glycerol release was similar at the two sites. The subcutaneous and omental alpha-adrenoceptors had similar affinities to 3H-RX821002 and showed characteristics of the alpha 2A subtype. The receptor densities were 220 +/- 21 and 460 +/- 84 fmol/mg of protein (means +/- s.e.m.) in omental and subcutaneous membranes, respectively. CONCLUSION: Inhibition of cyclic AMP accumulation and lipolysis by alpha 2A-adrenoceptors is less pronounced in omental than subcutaneous adipocytes which could be due to differences in receptor number. These differences in alpha 2A-adrenergic regulation could be of value in the treatment of the metabolic syndrome.

Beta-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 beta-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 beta-adrenergic receptor density and with the ratio of beta-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.

Cyclic AMP and glycerol concentrations in freeze-clamped human omental and subcutaneous adipose tissues.

OBJECTIVE: To compare intracellular concentrations of cyclic AMP, GTP, ATP, AMP and glycerol in omental and abdominal subcutaneous adipose tissues. DESIGN: Samples of omental and/or abdominal subcutaneous adipose tissues of 14 women undergoing elective surgery were freeze-clamped, deproteinized with perchloric acid and neutralized. All subjects were obese since it was not possible to freeze-clamp lean individuals. MEASUREMENTS: The concentrations of ATP and GTP were determined by ion-paired high performance liquid chromatography and AMP and glycerol enzymatically. Cyclic AMP was determined by radioimmunoassay. Intracellular nucleotide concentrations were calculated from tissue weights, water contents and relative amounts of sodium and potassium. RESULTS: The intracellular concentration of cyclic AMP in omental adipose tissue (31 mumol/l) was twice as high as that in subcutaneous fat while those of ATP, GTP and AMP were similar at the two sites. Glycerol concentrations were higher in subcutaneous (472 mumol/l) than in omental (325 mumol/l) adipose tissue. CONCLUSION: The results suggest higher relative stimulation of lipolysis by cyclic AMP in omental adipose tissue than in the subcutaneous region in situ. However, probably because of differences in blood flow and possibly in the lipolytic machinery, this is not reflected as a higher glycerol concentration.

Low particulate type IV phosphodiesterase activity in hypothyroid rat atria.

Rats were made hypothyroid by adding propylthiouracil (PTU) to their drinking water. Some of the PTU-treated rats were given thyroid hormone injections for 5 days. Both soluble and particulate cAMP-phosphodiesterase activities of adipose and ventricular tissues were increased by 25-60% in hypothyroidism. In left atria, soluble cAMP-phosphodiesterase activity was not significantly altered in hypothyroidism, while total particulate cAMP-phosphodiesterase activity was lowered by 30%. This lowering was due to diminished isoenzyme IV activity, as studied with the isoenzyme-specific inhibitors rolipram and SK&F 94836. In conclusion, the present results show decreased particulate type IV cAMP-phosphodiesterase activity in hypothyroid rat atria. This may explain the increased responsiveness to isoproterenol in hypothyroid atria.

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 alpha 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.

Defective stimulation of adipocyte adenylate cyclase, blunted lipolysis, and obesity in pseudohypoparathyroidism 1a.

Adipocyte plasma membranes were isolated from four patients with type 1a pseudohypoparathyroidism, a disease in which deficiency of the stimulatory guanine nucleotide binding protein Gs has been reported, and from controls. Stimulation of adenylate cyclase by isoproterenol was defective, whereas inhibition of forskolin-stimulated cyclase activity by N6-(phenylisopropyl)adenosine was normal. The patients had low serum FFA concentrations and developed obesity in childhood. These results suggest that pseudohypoparathyroidism 1a is connected with a blunted stimulatory response of adenylate cyclase, possibly because of low Gs activity, and that this blunted response may lead to decreased lipolysis and to obesity.

Enhanced negative inotropic effect of an adenosine A1-receptor agonist in rat left atria in hypothyroidism.

Left atria were isolated from rats made hypothyroid by adding propylthiouracil to their drinking water, such rats after saturating doses of thyroid hormones, and from control rats. Isoproterenol (ISO; 1 microM) increased the values of developed tension (DT), maximal rate of tension development (+dt/dt) and tension fall (-dT/dt). The effect was largest in hypothyroid and lowest in hyperthyroid atria. The adenosine A1-receptor agonist N6-(phenylisopropyl)-adenosine (PIA) had a powerful negative inotropic effect in ISO-stimulated atria. The effects of PIA on +dT/dt, -dT/dt and DT were enhanced in hypothyroidism. Adenosine receptor number was not decreased. The amount of total Gi-like proteins was estimated by pertussis toxin labeling. The amounts of Gi2 and Gi3 were estimated in Western blots using such antisera raised in rabbits against peptides corresponding to parts of their sequences, using purified recombinant alpha subunits as standards. The amounts of low and high molecular weight forms of Gs were estimated by cholera toxin labeling Gi2, Gi3 and pertussis toxin substrate concentrations were slightly lower in the hypothyroid animals, while the amounts of both forms of Gs per mg of protein were only half of those in euthyroid rat atria. The levels of Gi2 and Gi3 were greatly elevated as compared to Gs as membrane marker. These changes were reversed by treatment of the hypothyroid rats with thyroid hormones. In conclusion, the present results show an enhanced negative inotropic effect of an adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

A possible role for protein kinase C in the regulatory differences between intra-abdominal and subcutaneous human adipose tissue.

1. The adenosine A1-receptor agonist N6-(phenylisopropyl)adenosine inhibited lipolysis in a dose-dependent manner in human adipocytes. The effect is mediated by the inhibitory guanine-nucleotide-binding protein(s) that can be phosphorylated and thereby inactivated by protein kinase C. 2. Stimulation of protein kinase C by 12-O-tetradecanoyl-phorbol-13-acetate attenuated the inhibitory effect of the adenosine agonist. 3. Omental fat cells are less sensitive to adenosine than subcutaneous cells, although the stimulatory arm of cyclase regulation appears normal. Protein kinase C activity was measured in the soluble and particulate fractions of human omental and subcutaneous adipose tissue. Omental adipose tissue had a twofold higher membrane-bound and a threefold higher soluble protein kinase C activity. 4. It is therefore possible that the differences in regulation between the two sites are caused by different C kinase activities, causing variable phosphorylation of the inhibitory guanine-nucleotide-binding protein(s).

Regulation of adenylate cyclase in plasma membranes of human intraabdominal and abdominal subcutaneous adipocytes.

Fat cells were isolated from human subcutaneous, omental, and mesenteric adipose tissue. Omental fat cells were the smallest (438 pL), subcutaneous cells were intermediate (494 pL), and mesenteric cells were the largest (600 pL). There was no difference in the stimulation of adenylate cyclase by isoproterenol in plasma membranes of adipocytes prepared from the three sites. N6-(phenylisopropyl)adenosine inhibited 7-deacetyl-6-(N-acetylglycyl)forskolin-stimulated adenylate cyclase activity more potently in subcutaneous than in intraabdominal (especially omental) fat cell membranes. Kd values of the adenosine A1 receptors for 1,3-[3H]dipropyl-8-cyclopentylxanthine were similar in the three fat depots, but the receptor number as calculated per milligram protein was lower in omental than in abdominal subcutaneous adipocytes. Differences in adipocyte size cannot explain regional differences in the regulation of adenylate cyclase.

Weight loss normalizes the inhibitory effect of N6-(phenylisopropyl)adenosine on lipolysis in fat cells of massively obese human subjects.

1. Fat cells were isolated from massively obese patients at or before gastric bypass, from other patients after normalization of body weight after gastric bypass or gastroplasty (post-bypass patients) and from control subjects of a stable normal body weight. 2. The inhibition of isoprenaline-stimulated lipolysis by N6-(phenylisopropyl)adenosine in the presence of adenosine deaminase was much attenuated in cells from the massively obese patients as compared with those from normal-weight control subjects, but was normal in cells from post-bypass patients. 3. Isolated fat cells of the massively obese patients were larger (913 +/- 197 pl, mean +/- SEM) than those of the normal-weight group (437 +/- 95 pl). The volume of cells from the post-bypass patients was only 125 +/- 49 pl, although the body mass index of this group was almost exactly the same as that of the normal-weight control subjects. 4. Although epidemiological studies have suggested that genetic factors are important in the development and maintenance of obesity, these results demonstrate that the changes observed in the inhibitory regulation of lipolysis in obesity are secondary.

Attenuated adenosine-sensitivity and decreased adenosine-receptor number in adipocyte plasma membranes in human obesity.

Fat-cells were isolated from patients of body-mass indices (BMIs) ranging from 17.9 to 83.9 kg/m2. Isoprenaline-stimulated cyclic AMP accumulation in cells prepared from obese subjects as compared with normal-weight subjects, was less sensitive to inhibition by the adenosine agonist N6-(phenylisopropyl)adenosine (PIA) (P = 0.047). The inhibition of 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino) butyryl]-forskolin-stimulated adenylate cyclase by PIA in the presence of adenosine deaminase was also much attenuated in crude plasma membranes of adipocytes prepared from massively obese patients as compared with lean controls (P = 0.0143). This difference was probably not due to different cell size, because adenylate cyclase of crude plasma membranes of large adipocytes was actually more sensitive to PIA than was adenylate cyclase of membranes of smaller fat-cells co-isolated from the same individual. The stimulatory effect of PIA on glucose uptake in the presence of adenosine deaminase was depressed in adipocytes prepared from obese subjects and correlated with BMI at r = -0.626 (P = 0.007) at 100 nM-PIA. The adenosine receptors were studied by using the adenosine antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine. The binding was rapid and proportional to protein concentration. There was no difference in the affinities of receptors in membranes of obese and normal-weight subjects; Kd values of all patients averaged 3.3 nM. Bmax values were 54 and 130 fmol/mg of protein in membranes prepared from seven obese and five control patients respectively. The Bmax values calculated per mg of protein correlated with BMI at r = -0.539 (P = 0.047). The adenosine content of adipose tissue was higher in obese than in control subjects. These results demonstrate an attenuated response of cyclic AMP accumulation, adenylate cyclase and glucose uptake to adenosine in fat-cells prepared from obese subjects, and suggest that this change is at least partly due to changes in the amount of adenosine receptors, but not their affinity. The decreased receptor number could be due to higher adenosine content. A higher adenosine concentration in adipose tissue could explain why lipolysis is inhibited in situ in obesity, and the desensitization could explain the diminished response to adenosine analogues in isolated fat-cells.

Different metabolic regulation by adenosine in omental and subcutaneous adipose tissue.

Adenosine content was higher in omental adipose tissue (0.91 +/- 0.13 nmol g-1 of wet weight; mean +/- S.E.M.) than in abdominal subcutaneous adipose tissue (0.42 +/- 0.08 nmol g-1 of wet weight) in rapidly frozen surgical biopsy samples taken from ten patients undergoing elective abdominal surgery. This difference was statistically significant (P less than 0.002). The sensitivity of isoprenaline-stimulated lipolysis to inhibition by N6-(phenylisopropyl)adenosine was studied in omental and abdominal subcutaneous adipocytes isolated from nine patients. The effect of this adenosine Ri-site agonist was less pronounced in omental than in abdominal subcutaneous adipocytes which could be due to a desensitization phenomenon. This difference was statistically significant (P = 0.012). The ratio of the inhibitory guanine nucleotide binding proteins Gi1 and Gi2 to the corresponding stimulatory protein Gs was the same in plasma membranes prepared from omental and abdominal subcutaneous adipocytes. In conclusion, in omental adipose tissue, adenosine content is higher and the response to this nucleoside is less pronounced than in subcutaneous adipocytes. This difference cannot be explained by a different (Gi1 + Gi2)/Gs ratio.

Adipocyte plasma-membrane Gi and Gs in insulinopenic diabetic patients.

Changes in the amounts and functions of G-proteins have been reported in diabetic rats. We determined the Gi/Gs ratio of adipocyte plasma membranes in six insulinopenic diabetic patients and matched controls by immunoblotting with antisera against synthetic peptides corresponding to regions of G-proteins. No consistent changes in the Gi/Gs ratio were observed. It is still possible that the functional status of the G-proteins may be altered in diabetes.

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.