Selective modification of insulin action in adipose tissue by hyperthyroidism

Adipose-tissue lipolysis (assessed from glycerol release) and glucose uptake were examined in parametrial and mesenteric adipocytes prepared from control or hyperthyroid rats in relation to changes in insulin sensitivity. Basal rates of lipolysis did not differ significantly between adipose-tissue depots. Lipolysis was maximally stimulated by noradrenaline at 1 microM, half-maximal anti-lipolytic effects of insulin were observed at approximately 11 microU/ ml insulin, and half-maximal stimulation of glucose uptake was observed at approximately 16 microU/ml insulin in adipocytes from both depots. Wortmannin caused a dose-dependent inhibition of the anti-lipolytic effect of insulin (150 microU/ml) on noradrenaline-stimulated lipolysis. Half-maximal effects of wortmannin were observed at 20-40 nM. The p70S6K inhibitor rapamycin and the mitogen-activated protein kinase kinase inhibitor PD098059 had no effects on noradrenaline-stimulated lipolysis. Hyperthyroidism increased basal rates of lipolysis and the maximal response of lipolysis to noradrenaline stimulation (3.1-fold, P < 0.001 and 2.1-fold, P < 0.05 respectively) in parametrial adipocytes. Hyperthyroidism markedly blunted the sensitivity of noradrenaline-stimulated lipolysis to half-maximal suppression by insulin in both parametrial and mesenteric adipocyte depots, and noradrenaline-stimulated lipolysis at a maximal insulin concentration remained significantly higher in adipocytes prepared from hyperthyroid rats compared with controls. Hyperthyroidism had no effect on basal and little effect on insulin-stimulated glucose uptake. Tri-iodothyronine administered at a low dose selectively influenced the anti-lipolytic action of insulin in parametrial adipocytes, and led to significantly less marked elevation in plasma non-esterified fatty acid concentrations in vivo. The results demonstrate a selective effect of hyperthyroidism to impair insulin's anti-lipolytic action, and are consistent with the operation of different downstream signalling mechanisms for the effects of insulin on adipocyte glucose transport and lipolysis.     

Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth

Adipose tissue growth results from de novo adipocyte recruitment (hyperplasia) and increased size of preexisting adipocytes. Adipocyte hyperplasia accounts for the severalfold increase in adipose tissue mass that occurs throughout life, yet the mechanism of adipocyte hyperplasia is unknown. We studied the potential of macrophage colony-stimulating factor (MCSF) to mediate adipocyte hyperplasia because of the profound effects MCSF exerts on pluripotent cell recruitment and differentiation in other tissues. We found that MCSF mRNA and protein were expressed by human adipocytes and that adipocyte MCSF expression was upregulated in rapidly growing adipose tissue that encircled acutely inflamed bowel and in adipose tissue from humans gaining weight (4-7 kg) with overfeeding. Localized overexpression of adipocyte MCSF was then induced in rabbit subcutaneous adipose tissue in vivo using adenoviral-mediated gene transfer. Successful overexpression of MCSF was associated with 16-fold increases in adipose tissue growth compared with a control adenovirus expressing beta-galactosidase. This occurred in the absence of increased cell size and in the presence of increased nuclear staining for MIB-1, a marker of proliferation. We conclude that MCSF participates in adipocyte hyperplasia and the physiological regulation of adipose tissue growth.     

Leptin concentration in women is influenced by regional distribution of adipose tissue

Leptin concentrations in humans are increased with obesity, and women have higher leptin concentrations than men. This sex difference reflects the greater fat mass of women. However, there is evidence that factors other than the size of the adipose tissue mass contribute to serum leptin concentrations. This study was undertaken to determine whether anthropometric factors influenced leptin concentrations in our population. Leptin concentrations were measured in 375 persons from a population study of hypertension and diabetes for whom body-composition data (bio-electrical impedance analysis and anthropometry) were available. Serum leptin concentrations were more than four times higher in women than in men (18.5 +/- 13.9 compared with 3.8 +/- 3.6 ng/L, P < 0.0001). In individuals with comparable body mass indexes, these differences persisted after adjustment for either percentage fat (P < 0.05) or fat mass (P < 0.0001) by multivariate-regression analysis. After fat mass was adjusted for, the serum leptin concentration in both men and women was independent of waist circumference but in women was associated with hip circumference. Hip circumference is a proxy measure of peripheral fat and these results suggest that the larger hips of women may contribute to the sex difference in serum leptin concentration.     

Intra-abdominal adipose tissue in young children

OBJECTIVE: Intra-abdominal adipose tissue (IAAT) is associated with the metabolic complications of obesity. However the time course for the development of IAAT is not clearly defined because it is generally difficult to measure directly. The purposes of this short communication are to present data supporting the existence of IAAT in young children using direct measurement with computed tomography imaging, and to examine the relationship between IAAT and anthropometric indices in 16 healthy children (6.4 +/- 1.2 years; 24.8 +/- 5.4 kg). DESIGN: Total body fat (6.4 +/- 3.5 kg) and fat free mass (18.4 +/- 3.6 kg) were determined by bioelectrical resistance. Fat distribution was estimated from eight individual skinfold measurements, the ratio of three trunk skinfolds to three extremity skinfolds (0.78 +/- 0.20), and the waist:hip ratio (0.90 +/- 0.08). RESULTS: Mean abdominal subcutaneous adipose tissue (SCAT) was 65.3 +/- 44.8 cm2, and mean IAAT was 8.3 +/- 5.8 cm2. The ratio of IAAT to SCAT was 0.15 +/- 0.08, and the ratio of IAAT to total body fat was 1.44 +/- 0.84 cm2/kg. IAAT was significantly correlated with body weight (r = 0.54; P = 0.03), all skinfold measures (range r = 0.60-0.78; P = 0.02 to 0.0003) except at the calf, fat mass (r = 0.69; P = 0.003), and the trunk to extremity skinfold ratio (r = 0.78; P = 0.0003). There was no significant correlation between IAAT and the waist:hip ratio (r = 0.21). CONCLUSIONS: These preliminary results establish the existence of IAAT in young children and suggest that individual trunk skinfold measurements and the trunk:extremity skinfold ratio provide a better indication of IAAT compared to the waist:hip ratio. However, as with adults, the relationship between intra-abdominal adipose tissue and anthropometry in children is complex.     

Chemical sympathectomy of interscapular brown adipose tissue

Adult non-cold adapted rats were injected with 6-hydroxydopamine (6-OHDA) or saline and their interscapular brown adipose tissue (BAT) was removed after appropriate periods of time. Fluorescence histochemistry of control BAT demonstrated the presence of an extensive network of varicose fibers among the adipocytes and at the blood vessels. This was confirmed by electron microscopy which also revealed large and small dense core vesicles sparsely distributed in axons and terminals indicating the presence of noradrenaline (NA). After 6-OHDA injection the fluorescence from varicosities was abolished both among the adipocytes and at the vessels. Thus, chemical sympathectomy was more effective than surgical- or immunosympathectomy, which spare the innervation of adipocytes. Parallelling the disappearance of fluorescence was a significant decrease of measurable NA. During recovery the extractable NA increased before the reappearance of fluorescent varicosities. This could be explained by transmitter accumulation in the nervetrunks within the tissue, which, in general, appeared unaffected by 6-OHDA. A large number of cells with a strong yellowish fluorescence distributed through the BAT was unaffected by 6-OHDA. There was no evidence for the presence of intrinsic ganglia.     

RU486 is a potent inhibitor of aromatase induction in human breast adipose tissue stromal cells

Aromatization of circulating androgens in adipose tissue is a major source of estrogens in postmenopausal women. As part of our efforts to elucidate the mechanism of aromatase induction in human breast adipose tissue, we tested the effects of the antiglucocorticoid and antiprogestin, RU486, on aromatase induction in primary cultures of adipose tissue stromal cells in a serum-free system devoid of phenol-red. Under these conditions 1 microM cortisol alone induces low levels of aromatase activity within one day, whereas platelet-derived growth factor BB (PDGF) potentiates this effect two- to three-fold. The well-known strong inductive effect of dibutyryl-cAMP (db-cAMP) is also augmented by cortisol, but is inhibited by PDGF in the absence of cortisol. RU486 completely prevented aromatase induction by cortisol and PDGF. Induction by db-cAMP in the presence and absence of cortisol was significantly inhibited by RU486. Even lower activities were measured when RU486 was added to cells stimulated with PDGF and db-cAMP in the absence or presence of cortisol. Similar results were obtained after prolonged incubation. The inhibitory effects of RU486 are dose dependent, less than 1 microM completely blocking the effects of cortisol, whereas 10 microM are needed to block db-cAMP induction. RU486 does not affect cell number, cellular protein, viability or house-keeping enzymes such as lactate dehydrogenase (LDH), and therefore seems to act specifically. The time course of RU486 action on the db-cAMP induction of aromatase indicates that it acts via a newly synthetized mediator or target in stromal cells. These results suggest that all known inducers of aromatase in adipose tissue depend upon the action of signalling molecules (probably members of the nuclear receptor superfamily) which can be blocked by RU486. The inhibitory action of PDGF seems to be independent of steroid hormone action, as seems some basal activity induced by db-cAMP. In conclusion, this in vitro study suggests that RU486 might be a useful tool for the therapy of estrogen-dependent tumours through its inhibition of aromatase induction.     

Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women

Insulin action and GLUT4 expression were examined in adipose tissue of severely obese premenopausal women undergoing gastrointestinal surgery. Fat samples were taken from three different anatomical regions: the subcutaneous abdominal site, the round ligament (deep abdominal properitoneal fat), and the greater omentum (deep abdominal intraperitoneal fat). The stimulatory effect of insulin on glucose transport and the ability of the hormone to inhibit lipolysis were determined in adipocytes isolated from these three adipose depots. Insulin stimulated glucose transport 2-3 times over basal rates in all adipocytes. However, round ligament adipose cells showed a significantly greater responsiveness to insulin when compared to subcutaneous and omental adipocytes. Round ligament fat cells also displayed the greatest sensitivity and maximal antilipolytic response to insulin. We also investigated whether regional differences in fat cell insulin-stimulated glucose transport were linked to a differential expression of the GLUT4 glucose transporter. GLUT4 protein content in total membranes was 5 and 2.2 times greater in round ligament adipose tissue than in subcutaneous and omental fat depots, respectively. Moreover, GLUT4 mRNA levels were 2.1 and 3 times higher in round ligament than in subcutaneous or omental adipose tissues, respectively. Adipose tissue GLUT4 protein content was strongly and negatively associated (r = -0.79 to -0.89, p < 0.01) with the waist-to-hip ratio but not with total adiposity. In conclusion, these results demonstrate the existence of site differences in adipose tissue insulin action in morbidly obese women. The greater insulin effect on glucose transport in round ligament adipocytes was associated with a higher expression of GLUT4 when compared to subcutaneous abdominal and omental fat cells. Moreover, despite the regional variation in GLUT4 expression, an increased proportion of abdominal fat was found to be associated with lower levels of GLUT4 in all adipose regions investigated.     

Uncoupling proteins: beyond brown adipose tissue

Uncoupling protein, originally described in the inner mitochondrial membrane of brown adipose tissue, permits the oxidation of fuels without the generation of adenosine triphosphate (ATP). Closely related proteins have now been found in many other tissues and shown to be regulated by thyroid hormones and dietary factors. These uncoupling proteins may play a significant role in energy expenditure, with implications for the development of human obesity.     

Differential mobilization of fatty acids from adipose tissue

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells. While VEGF-C is homologous to other members of the VEGF/platelet derived growth factor (PDGF) family, its C-terminal half contains extra cysteine-rich motifs characteristic of a protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. VEGF-C is proteolytically processed, binds Flt4, which we rename as VEGFR-3 and induces tyrosine autophosphorylation of VEGFR-3 and VEGFR-2. In addition, VEGF-C stimulated the migration of bovine capillary endothelial cells in collagen gel. VEGF-C is thus a novel regulator of endothelia, and its effects may extend beyond the lymphatic system, where Flt4 is expressed.     

Intrinsic regulation of blood flow in adipose tissue

Previous studies on intact human subcutaneous tissue have shown, that blood flow remains constant during minor changes in perfusion pressure. This so-called autoregulatory response has not been demonstrable in isolated preparations of adipose tissue. In the present study on isolated, denervated subcutaneous tissue in female rabbits only 2 of 12 expts. revealed an autoregulatory response during reduction in arterial perfusion pressure. Effluent blood flow from the tissue in the control state was 15.5 ml/100 g-min (S.D. 6.4, n = 12) corresponding to slight vasodilatation of the exposed tissue. Following total ischemia all experiments showed a period with reactive hyperemia, and both duration of hyperemia and excess flow was related to the duration of the ischemia. This response therefore seems more resistant to the experimental procedure, while autoregulation of blood flow to lowered pressure is more susceptible to surgical exposure of the tissue. During elevation of arterial perfusion pressure blood flow in the isolated tissue showed a transient increase and then almost returned to the level during normotension, indicating an elevated vascular resistance. Raising of venous pressure elicited vasoconstriction with pronounced flow reduction. These two reactions may be important for local regulation of blood flow in subcutaneous tissue during orthostatic changes in arterial and venous pressure. It is concluded that the response in adipose tissue to changes in arterial pressure (autoregulation), venous pressure and total ischemia appear to be elicited by different mechanisms.     

Lipoprotein and hormone-sensitive lipases in porcine adipose tissue

Lipoprotein lipase (LPL) is an adipocyte enzyme that cleaves fatty acids from circulating lipoproteins. Fatty acids enter the cell to be oxidized or esterified. Hormone-sensitive lipase (HSL) is an adipocyte enzyme that cleaves fatty acids from intracellular triacylglycerol. The HSL is activated by phosphorylation. Assays for the two lipases are complex because the hydrophobic substrate, triacylglycerol, must be presented as a gum-based suspension or as a detergent-based emulsion to a relatively hydrophilic enzyme. A convenient, stable glycerol/phospholipid suspension of the substrate was used for measurement of porcine adipose tissue LPL and HSL in vitro. This substrate was excellent for LPL. It produced rates five times those using a more complex and less convenient gum-based substrate suspension. The LPL activity was released by heparin, had a pH optimum of approximately 8.5, was activated by serum, and was inhibited by NaCl and protamine. This LPL assay measures enzyme capacity. The same substrate was used to measure an adipose tissue lipase activity that had a pH optimum below 7, was not activated by serum, and was not inhibited by NaCl or protamine. These are all characteristics of HSL. Despite the convenience, this substrate was not appropriate for porcine adipose tissue HSL because the rates were only 30 to 50% of those produced with a more complex, less convenient gum-based substrate suspension. Furthermore, incubation of enzyme or tissue slices with insulin, or agents that elevate cAMP concentration, did not modulate this lipase activity, as expected. These incubations poorly modulated LPL activity.     

Adrenergic receptors mediating depolarization in brown adipose tissue

Adrenergic receptors mediating depolarization in in vitro neonatal rat brown adipose tissue (BAT) have been characterized by use of adrenergic agonists and antagonists. Releasable endogenous catecholamine was present in BAT as demonstrated by tyramine- and 1,1-dimethyl-4-phenylpiperazinium iodide- (DMPP) induced depolarization in BAT from normal rats and its absence when BAT from reserpinized rats was used. In BAT from reserpinized rats l-norepinephrine, l-phenylephrine, and l-isoproterenol all similarly depolarized the bronw adipocytes over the concentration range of 10(-8) to 10(-6) M with a maximal depolarization of about 25 mV. Dopamine and d-norepinephrine were more than 100 times less potent. The beta-adrenergic blocker propranolol competitively inhibited isoproterenol-induced depolarization, whereas the alpha-adrenergic blackers, phentolamine and phenoxybenzamine, inhibited the phenylephrine-induced depolarization with much smaller inhibitory effects on the isoproterenol-induced depolarization. Both phenylephrine and isoproterenol elicited transient depolarizations when briefly added to the bathing medium while continuously recording from the same cell. Both the agonist and antagonist studies are interpreted as indicating the presence of both alpha- and beta-adrenergic receptors on BAT cells which mediate catecholamine-induced depolarization.     

Modulation of white adipose tissue lipolysis by nitric oxide

In isolated adipocytes, the nitrosothiols S-nitroso-N-acetyl-penicillamine (SNAP) and S-nitrosoglutathione stimulate basal lipolysis, whereas the nitric oxide (NO.) donor 1-propamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA-NONOate) or NO gas have no effect. The increase in basal lipolysis due to nitrosothiols was prevented by dithiothreitol but not by a guanylate cyclase inhibitor. In addition the cyclic GMP-inhibited low Km, cyclic AMP phosphodiesterase activity was inhibited by SNAP suggesting that SNAP acting as NO+ donor increases basal lipolysis through a S-nitrosylation mediated inhibition of phosphodiesterase. Contrasting with these findings, SNAP reduced both isoproterenol-stimulated lipolysis and cyclic AMP production, whereas it failed to modify forskolin-, dibutyryl cyclic AMP-, or isobutylmethylxanthine-stimulated lipolysis, suggesting that SNAP interferes with the beta-adrenergic signal transduction pathway upstream the adenylate cyclase. In contrast with SNAP, PAPA-NONOate or NO gas inhibited stimulated lipolysis whatever the stimulating agents used without altering cyclic AMP production. Moreover PAPA-NONOate slightly reduces (30%) the hormone-sensitive lipase (HSL) activity indicating that stimulated lipolysis inhibition by NO. is linked to both inhibition of the HSL activity and the cyclic AMP-dependent activation of HSL. These data suggest that NO. or related redox species like NO+/NO- are potential regulators of lipolysis through distinct mechanisms.     

Catabolism of adipose tissue by a tumour-produced lipid-mobilising factor

Induction of lipolysis in murine white adipocytes by a tumour lipid-mobilising factor (LMF) was associated with stimulation of adenylate cyclase in adipocyte plasma membrane preparations. Induction of lipolysis was attenuated by the adenylate cyclase inhibitor MDL12330A and the protein kinase A inhibitor H8, suggesting that cAMP was the intracellular mediator of induction. The effect of LMF on adenylate cyclase was responsive to GTP, with low concentrations (0.1 microM) causing stimulation and high concentrations (10 microM) causing inhibition, suggesting the involvement of both stimulatory (Gs) and inhibitory (Gi) guanine nucleotide-binding proteins. At a concentration of 10 microM, propranolol noncompetitively reduced the induction of lipolysis by LMF. Thus, lipolysis in white adipose tissue during the process of cancer cachexia is mediated by a tumour factor which stimulates cAMP production, possibly through a beta-adrenergic receptor.     

Impaired regulation of adipose tissue lipoprotein lipase in obesity

Among the numerous alterations in adipose tissue metabolism in obesity is an increased activity of lipoprotein lipase (LPL), the rate-limiting enzyme in triglyceride assimilation. The present paper summarizes a series of experiments demonstrating that the regulation of LPL activity is also impaired in obesity. In normal subjects, glucose intake leads to an increase in adipose tissue LPL activity; this phenomenon can also be demonstrated by incubation of adipose tissue in an insulin-containing buffer in vitro. In contrast, the initially high enzyme activity of adipose tissue from obese subjects is non-responsive to glucose and insulin in vivo and in vitro. However, incubation of adipose tissue from obese subjects in the absence of glucose and insulin led to a rapid decline in enzyme activity and elicited a normal response to subsequent exposure to glucose and insulin. These data indicate that the hyperinsulinemia of the obese tissue donor may be of importance for the abnormalities of LPL activity. However, experiments with VMH-lesioned rats demonstrated that insulin levels and LPL activities could be dissociated. In obese and normal-weight rats, standardized with regard to insulin levels by abolition of endogenous insulin production by streptozotocin treatment and substitution with identical doses of insulin, LPL activities and regulation differed markedly, indicating that factors other than insulin are also involved in the dysregulation of LPL activity in obesity.     

Kinetic properties of ovine adipose tissue fructose-1,6-bisphosphatase

The presence of FBPase was confirmed in both human and ovine white adipose tissue in metabolically significant amounts. The partially purified enzyme from ovine adipose tissue exhibited kinetic properties very similar to other mammalian FBPases (pH optimum of 7.5, absolute requirement for divalent metal ions and strong inhibition by both AMP and F-2,6-P2). The micromolar S0.5 value obtained suggests that the enzyme may be of physiological significance.