Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic.

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

Anthropometric indexes for visceral fat estimation in overweight/obese women attending to age and menopausal status.

The objective of the present study was to assess the usefulness and accuracy of different anthropometric measurements in the diagnosis of abdominal visceral obesity in overweight/obese women attending to age and menopausal status. The secondary objective was to evaluate the usefulness of waist circumference (WC) in two different sites. Different anthropometric indicators were assessed in 55 overweight/obese women (n=22 premenopausal, n=33 postmenopausal; BMI > 25 kg/m(2)) and compared with computed tomography measurements of abdominal visceral adipose tissue (VA) performed as a single scan at L4-L5. Our results show that VA significantly differs between both groups of women. Waist2-hip ratio (W2HR) was significantly correlated to VA in both groups of women. After multiple regression analysis, sagittal diameter was an independent parameter to predict VA. However, no significant differences were obtained in this diameter between both groups. None of the waist circumferences were significantly associated to VA. Moreover, the two sites of WC were statistically different. In conclusion, waist-to-hip ratio, measured immediately above the iliac crest (W2HR) seems to be the more appropriated anthropometric index for the estimation of visceral fat in women, independently of age. Although sagittal diameter is a fine parameter to predict visceral fat area, it is not adequate to discriminate between women of different age or menopausal status. In contrast with previous findings, waist circumferences do not seem to be useful for predicting VA.

Role of heterotrimeric G-proteins in epidermal growth factor signalling.

Since in 1986 it was reported that a pertussis toxin-sensitive substrate was involved in the Ca2+ signal induced by epidermal growth factor (EGF) in rat hepatocytes, much evidence accumulated to implicate heterotrimeric G-proteins in EGF action. EGF can also induce a cyclic AMP signal, but while the generation of a Ca2+ signal appears to be quite general in EGF action, the increase in cyclic AMP occurs only in few cell types. In non-transformed cell types these effects appear to involve G-proteins. EGF not only induces cell proliferation but also interacts with hormones in the short-term control of cell function in quiescent cells. Most of the known interactions are on cyclic AMP mediated hormone effects, and in many cases, the interaction between EGF and hormones involves G-proteins. Here we review the evidence accumulated in recent years that implicate G-proteins in EGF action. An understanding of the mechanisms involved may reveal new mechanisms of G-protein regulation and will contribute to our knowledge of EGF function and signal transduction.

The antilipolytic effects of insulin and epidermal growth factor in rat adipocytes are mediated by different mechanisms.

Epidermal growth factor (EGF) and insulin induced similar effects in isolated rat adipocytes. To determine whether EGF and insulin produced similar effects through the same mechanisms, we focused on lipolysis. Insulin inhibited the lipolysis stimulated by isoproterenol, glucagon (either alone or in combination with adenosine deaminase), adenosine deaminase itself, or forskolin. In contrast, EGF did not inhibit the lipolysis stimulated by forskolin or by hormones when the cells were also incubated with adenosine deaminase. The effect of insulin, but not that of EGF, on isoproterenol-stimulated lipolysis disappeared when adipocytes were incubated with 1 microM wortmannin. These results indicate that EGF and insulin affected lipolysis through different mechanisms. We observed that EGF, but not insulin, increased cytosolic Ca2+. The effect of EGF, but not that of insulin, disappeared when the cells were incubated in a Ca2+-free medium. We suggest that EGF, but not insulin, mediate its antilipolytic effect through a Ca2+-dependent mechanism which, however, do not involve Ca2+-activated protein kinase C isoforms. This is based on the following: 1) phorbol 12-myristate 13-acetate affected lipolysis in an opposite way to that of EGF; and 2) the protein kinase C inhibitor bisindolylmaleimide GF 109203X did not affect the antilipolytic action of EGF. Our results indicate that the antilipolytic effect of EGF resembles more that of vasopressin than that of insulin.

Epidermal growth factor secreted from submandibular salivary glands interferes with the lipolytic effect of adrenaline in mice.

We had described that epidermal growth factor (EGF) interfered with the lipolytic effect of catecholamines in isolated adipocytes. Since catecholamines stimulate the release of EGF from submandibular salivary glands to blood plasma in male mice, we studied whether EGF affected also the lipolytic response to adrenaline in whole animals. We studied the effect of adrenaline in sialoadenectomized and sham-operated mice receiving or not a high dose of EGF following adrenaline injection. There was no difference in plasma EGF concentration between sham-operated and sialoadenectomized animals receiving saline. After adrenaline administration plasma EGF increased by 20-fold in sham-operated but did not increase in sialoadenectomized mice. Indeed, the increase was much higher (more than 100-fold) in mice receiving exogenous EGF. The effect of adrenaline on plasma concentration of both glycerol and nonesterified fatty acids was higher as lower was plasma EGF concentration. Isolated adipocytes obtained from sham-operated or sialoadenectomized mice had identical lipolytic response to adrenaline. The lipolytic response of adipocytes to isoproterenol was decreased by addition of EGF. To study whether the interference with the in vivo lipolytic effect of adrenaline had further metabolic consequences, we measured plasma beta-hydroxybutyrate concentration in plasma. There was no difference in the response to adrenaline between sham-operated and sialoadenectomized mice in spite of the difference in plasma nonsterified fatty acid concentration. Studies in isolated hepatocytes indicated that ketogenesis run at near maximal rate in this range of substrate concentration. These results suggest that EGF in the physiological range decreases the lipolytic effect of adrenaline but does not compromise further metabolic events like the enhancement of ketogenesis.

Activation of Raf-1 is defective in annexin 6 overexpressing Chinese hamster ovary cells.

Annexin 6 is a Ca2+-dependent phospholipid-binding protein involved in membrane trafficking. In this study we demonstrate the association of Raf-1 with recombinant rat annexin 6. Raf-annexin 6 interaction was shown to be independent of cell activation by epidermal growth factor (EGF) or phorbol esters (12-O-tetradecanoyl-phorbol-13-acetate (TPA)). A stable Chinese hamster ovary (CHO)-anx6 cell line overexpressing annexin 6 was established to examine the function of annexin 6. In these cells, no increase of Ras-GTP levels, induced by EGF or TPA, was detected. In addition, the activity of Raf was completely inhibited, whereas the mitogen-activated protein kinase-P was unaffected.

Site-specific differences in the fatty acid composition of abdominal adipose tissue in an obese population from a Mediterranean area: relation with dietary fatty acids, plasma lipid profile, serum insulin, and central obesity.

BACKGROUND: Abdominal obesity is associated with coronary risk, although causality is not well established. OBJECTIVE: In an obese Mediterranean population, we measured the fatty acid composition of adipose tissue, its relation with dietary fatty acids and central fat deposition, and its influence on plasma lipids and insulin. DESIGN: Adipose tissue samples were obtained from 84 obese patients (29 men, 55 women) aged 30-70 y (body mass index, in kg/m(2): 27-35). We measured concentrations of insulin and lipids in plasma and fatty acids in subcutaneous, omental, and perivisceral fat. Dietary fatty acid intake was assessed with a 7-d diet record. RESULTS: The population studied was normolipidemic and normoinsulinemic. There were important differences in fatty acid composition between tissue sites: saturated fatty acids were higher and monounsaturated fatty acids were lower in perivisceral than in subcutaneous fat (P < 0.05). Significant correlations were found for oleic, linoleic, alpha-linolenic, and total n-6 polyunsaturated fatty acids between the subject's habitual diet and adipose tissue composition. Oleic and n-3 fatty acids from adipose regions were negatively correlated with apolipoprotein B and triacylglycerols; adipose tissue 22:1n-9, 20:2n-6, stearic acid, and eicosapentaenoic acid were positively correlated with HDL and apolipoprotein A; and adipose tissue myristic acid was negatively correlated with apolipoprotein A (P < 0.05). Central obesity was positively associated with n-6 polyunsaturated fatty acids and inversely associated with monounsaturated fatty acids and n-3 polyunsaturated fatty acids in adipose tissue (P < 0.05). CONCLUSION: The differences found in the composition and metabolism of perivisceral, omental, and subcutaneous fats may indicate that their atherogenic capacities also differ.

Anthropometric, computed tomography and fat cell data in an obese population: relationship with insulin, leptin, tumor necrosis factor-alpha, sex hormone-binding globulin and sex hormones.

OBJECTIVE: To correlate anthropometric, computed tomography and fat cell data from abdominal regions with the levels of serum insulin, C-peptide, leptin, tumor necrosis factor-alpha (TNF-alpha), testosterone, 17beta-estradiol, androstenedione, dehydroepiandrosterone sulphate (DHEA-S) and sex hormone-binding globulin (SHBG). DESIGN AND METHODS: The sample consisted of 84 obese patients (29 men, 22 premenopausal women and 33 postmenopausal women) who had undergone abdominal surgery. Weight, height, percentage of body fat by skinfolds, waist, hip and thigh circumferences, sagittal and coronal diameters, visceral and subcutaneous area, serum hormones and fat cell data were studied. RESULTS AND CONCLUSIONS: Premenopausal women showed the lowest values in most abdominal distribution parameters, although, depending on the waist circumference criteria at the umbilicus level perimeter (W1) or midway between lower rib margin and iliac crest perimeter (W2), the population was classified differently, as gynoid or android. Although there were no differences in fat cell size between genders, gynoid women had smaller and more numerous fat cells than the android type. Perivisceral fat cell size was significantly smaller than subcutaneous fat cell size. In women, central obesity was significantly correlated with an increase in serum insulin, leptin, TNF-alpha, testosterone and androstenedione levels, and a decrease in 17beta-estradiol and DHEA-S, while in men significant correlations were positive with insulin and negative with testosterone and androstenedione. Fat cell size was positively correlated with serum levels of leptin, insulin, DHEA-S, androstenedione and inversely correlated with SHBG. These data indicate that hormones seem to interact not only with body fat distribution but also with fat cell size. This interaction differs between genders and between the different abdominal adipose tissue regions.

Efficacy of preoperative diagnostic imaging localization of technetium 99m-sestamibi scintigraphy in hyperparathyroidism.

BACKGROUND: Until now, preoperative parathyroid imaging has been considered unnecessary because currently available techniques do not provide any better results than an expert surgeon. We conducted a multicenter prospective study evaluating the capability of technetium 99m sestamibi scintigraphy in the preoperative identification of pathologic glands. METHODS: Ninety-three patients with hyperparathyroidism, seven of them reoperative cases, were studied. In addition, 20 age-matched normocalcemic control subjects were also studied. RESULTS: Surgical confirmation of scintigraphic images was obtained in 91 of 93 cases (sensitivity, 97.8%). In all reoperative cases (n = 7), in all cases of ectopic glands (whether operated or not; n = 6), and in all patients with a single-gland disease (n = 70), topographic identification of the pathologic gland was correct in 100%. In multiple-gland disease (n = 23), involvement of more than one gland was visualized in only 61% of the patients; however, in 91% of patients, at least one gland was localized. Surgical success-defined as postoperative normocalcemia-with this approach was 100%. All scans of normocalcemic control subjects were negative. Of 31 patients in whom a multinodular goiter coexisted, seven presented a significant radionuclide background at 120 minutes' scan. False-positive images were found together with those corresponding to the pathologic glands in only three cases. CONCLUSIONS: 99mTc-sestamibi is a highly reliable, sensitive, and specific technique for imaging of pathologic glands in hyperparathyroidism, especially in single-gland disease. It may be considered as a first line single-procedure when a preoperative topographic diagnosis is required.

Papillary thyroid carcinoma: prognostic index for survival including the histological variety.

BACKGROUND: Numerous prognostic factors have been studied for survival in patients with papillary thyroid carcinoma (PTC), although there are few multivariate studies that include the histological variety of PTC. HYPOTHESIS: There are prognostic factors that influence survival in a series of patients with PTC, including the histological variety, and a new prognostic index (PI) for survival can be formulated by accounting for these factors. DESIGN: A retrospective study. SETTING: A university hospital department of surgery. PATIENTS: Between January 1970 and December 1995, 200 patients undergoing surgery for PTC were observed (mean follow-up, 8 years). MAIN OUTCOME MEASURES: A univariate analysis was done for survival rates using the Kaplan-Meier estimation method. The possible prognostic factors were evaluated using a multivariate analysis according to the Cox model. We formulated a PI and defined 3 risk groups (low, medium, and high) for mortality. RESULTS: Of the 200 patients, 175 (87.5%) are still alive. Of the 25 deaths, 19 (9.5%) were due to the tumor. The survival was 97.5% at 1 year, 92.8% at 5, 89.5% at 10, and 83.9% at 15 and 20 years. The prognostic factors obtained after the multivariate analysis were age, tumor size, extrathyroid spread, and histological variant of the PTC. The PI is calculated as follows: PI = (2 x size) + (6 x spread) + (2 x variant) + (3 x age). As for the risk groups, the low-risk group showed a mortality of 0%; the medium-risk group, 17.1%; and the high-risk group, 76.5%. CONCLUSIONS: The histological variety of PTC has prognostic value for survival in patients with PTC. As risk factors for PTC mortality, we consider an age of 50 years or older, a tumor larger than 4 cm, the existence of extrathyroid spread, and a certain histological subtype of PTC. With these risk factors, it is possible to formulate a PI and classify patients into low-, medium-, and high-risk groups for mortality.

Body fat distribution in pre-and post-menopausal women: metabolic and anthropometric variables.

BACKGROUND: Abdominal obesity is associated with coronary risk although the causality is not well established. OBJECTIVE: To compare body fat distribution and metabolic variables in obese pre- and post- menopausal women. DESIGN: The study was conducted in 55 obese female subjects (22 pre- and 33 post- menopausal) with body mass indices (BMI) from 27 to 35 Kg/m2. Body fat distribution was measured using waist and hip circumference and computerized tomography. Hormones such as estradiol, testosterone, androstenedione, dehydroepiandrosterone-sulfate, as well as plasma lipids such as triglycerides, total cholesterol, high density lipoprotein, low density lipoprotein, apoproteins A and B, were also determined. RESULTS: No significant differences were found between the two groups in BMI, percentage of body fat, waist circumference, triceps, biceps, suprailiac and abdominal skinfolds. Hip circumference, subscapular skinfold and subcutaneous abdominal adipose tissue area were significantly lower, and waist-hip ratio, visceral area and visceral to subcutaneous area ratios significantly higher in post- menopausal women (P < 0.05). As was expected, basal plasma estrogens, and dehydroepiandrosterone-sulfate, were significantly higher in pre-menopausal women. When comparing plasma lipids, blood cholesterol and low density lipoprotein, values were significantly higher in the post-menopausal group (P < 0.05). CONCLUSIONS: Our data suggest that body fat distribution changes according to menopausal status, with central obesity more pronounced in post- menopausal women. Metabolic abnormalities are related more to visceral adipose tissue and estrogen plasma levels than to BMI.

Interrelationship between serum lipid profile, serum hormones and other components of the metabolic syndrome.

The aim of the present study was to investigate the association between the serum lipid profile and components of the metabolic syndrome, such as central obesity (anthropometric, computed tomography and fat cell data), insulin, sex-hormone-binding-globulin (SHBG) and different hormones influencing this important syndrome, e.g. sex steroids, leptin and tumor necrosis factor-alpha (TNF-alpha). The sample consisted of 85 obese patients (30 men and 55 women) who had undergone abdominal surgery. Fasting serum lipids were analysed, as well as anthropometric and computed tomography data, perivisceral and subcutaneous fat cell size and serum glucose and hormones. Abdominal fat revealed itself as an important correlator of the adverse changes in plasma lipoprotein levels, the waist-to-hip-ratio and waist-to-thigh-ratio being the best morphological correlators in men and women, respectively. Intra-abdominal fat (VA) correlated significantly and positively to perivisceral fat cell size in women, while no correlation was found between subcutaneous fat accumulation (SA) and adipocyte size in both genders. Perivisceral fat cell size showed the greatest number of correlations with the adverse plasma lipid profile compared to that in the subcutaneous depot. SHBG and sex steroids showed a negative correlation with serum lipids considered a cardiovascular risk. In contrast, TNF-alpha and C-peptide were inversely correlated with potential protector lipids. In conclusion, abdominal obesity, adipocyte hypertrophy from visceral fat, serum TNF-alpha and C-peptide seem to be the best correlators of the lipoprotein disturbance characteristic of the metabolic syndrome, whereas SHBG and sex steroids could play a protective role regarding the lipid profile associated to this syndrome.

Adiposity and dietary intake in cardiovascular risk in an obese population from a Mediterranean area.

The aim of the study was to determine the particular relevance of android fat distribution and dietary intake in cardiovascular risk in an obese Mediterranean population with high intake of monounsaturated fatty acids (MUFA) and to compare the findings with those from normal-weight subjects. For the study, 193 subjects aged 25-60 were selected: 118 obese (BMI > or = 27 kg/m2), and 75 normal-weight (BMI < 25 kg/m2). Cardiovascular risk factors including hypertension, dyslipidaemia, glucose intolerance and insulin resistance were assessed. Nutrient intake and body fat distribution were determined. Results show that MUFA were highly consumed in the total population (21% of total energy). The obese population was normolipidemic and normoinsulinemic. However, cardiovascular risk factors (CVRF) were significantly higher than in normal-weight (P < 0.05). Obese subjects derived a greater percentage of their energy intake from total fat and lower from carbohydrates and saturated fats (P < 0.05). BMI and waist-hip ratio positively correlated with fat percentage of total energy intake and with MUFA (g/100 g fatty acids) in men, indicating that the excess of fat intake in obesity is due to a larger consumption of olive oil. CVRF were significantly and positively associated to waist circumference and WHR, both in obese and in normal-weight subjects. In conclusion, not only obesity but also android fat in normal-weight subjects are important factors in cardiovascular disease even in the Mediterranean population, with a high intake of MUFA, where these factors seem to be more relevant to cardiovascular risk than dietary composition.

[Evaluation of food consumption in overweight women before starting voluntary dieting programs]. / Evaluación del consumo alimentario en mujeres con sobrepeso que van a ser sometidas a un programa de adelgazamiento voluntario.

The dietary intake of 50 premenopausal, overweight women, aged between 18 and 50 years old (31 +/- 9 years), with a mean body mass index of 29.9 +/- 3.1 kg/m2, was evaluated using the 24 hour recall method, the day before the start of a voluntary dietetic treatment. All the women had repeatedly tried losing weight throughout the courses of their lives. Caloric restriction was compared with weight loss after a week of dietetic treatment. Our results indicate that none of the women taking part in the study reported diets balanced in energy and nutrients. Although in 50% of the patients dietary energy intake was insufficient, all of them, when submitted to a diet of 1200 kcal per day, lost more weight (0.8-1.5 kg) than expected. The high fat intake and above all carbohydrate deficiency in these patients, indicate the lack of nutritional knowledge in their weight loss attempts. In our opinion, nutritional education is necessary to help them implement a balanced diet.

[Infections in the diabetic. Comparative study of infections in the foot and other locations]. / Infecciones en el diabético. Estudio comparativo de las infecciones del pie y de otras localizaciones.

Given the high morbi-mortality of foot infections among the diabetics and the poor knowledge of their predictive, clinical and evolutive factors, we have retrospectively studied a group of patients with these characteristics, comparing them with infections among diabetics affecting other locations. We studied 66 infections among diabetics: 34 patients with diabetic's foot and 32 with infections at other locations: 20 pyelonephritis and 12 pneumonias. Medical records were obtained in all cases and all patients underwent a complete physical exploration in order to assess their risk factors. We observed as a significant predictive factor of diabetic's foot, diabetes type I, with an evolution longer than 10 years, neuropathy, vasculopathy or retinopathy. From the clinical point of view and compared with the other infections, these patients showed longer hospitalization, greater initial clinical severity, glucemias higher than 200 mgr/l., anemia and high GSR. Ethiologically, the infection of diabetic's foot was polymicrobian in 42.3% of all cases, being S. aureus the microorganism more frequently isolated. On the contrary, in infections at other locations, monomicrobian flora was more frequent, being E. coli the most frequent in pyelonephritis and S. pneumoniae in pneumonias. The evolution was satisfactory in all cases, with a close medical and surgical combined treatment and the appropriate use of antibiotic combinations, mainly clindamicine + tobramicine in the diabetic's foot and cefuroxime in the other locations.

MYH polyposis syndrome: clinical findings, genetics issues and management.

Colorectal cancer (CRC) is one of the most frequent cancer in first world. Two hereditary CCR syndrome have been described: familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer. A recently described biallelic mutation of MYH, is responsible for adenomatous polyposis with an increased risk of CRC and is responsible for 30-40 % of adenomatous polyposis cases in which an APC mutation cannot be found. However, there is no clear consensus in the literature as whether a monoallelic mutation increases the risk for colorectal cancer. In addition, some authors have indicated that the spectrum of extracolonic lesions in MYH associated polyposis (MAP) might be far different from that observed in FAP and could be more similar to Lynch syndrome spectrum. In this review we are going to describe some general and specific aspects of MAP, including genetic topics, clinical features, different phenotypes and strategies to reduce CCR risk.

Annexin A6 is a scaffold for PKCα to promote EGFR inactivation.

Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.

Annexin A6 inhibits Ras signalling in breast cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signalling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca(2+)- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca(2+)- and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.

Cortisol secretary pattern and glucocorticoid feedback sensitivity in women from a Mediterranean area: relationship with anthropometric characteristics, dietary intake and plasma fatty acid profile.

BACKGROUND: Chronic stress is associated with a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis consisting on disturbances on the cortisol response and lipid metabolism. OBJECTIVE: To evaluate the HPA axis activity in women from a Mediterranean area, comparing three different measurements: daily cortisol secretory variability, postprandial cortisol secretion and glucocorticoid feedback sensitivity. In addition, HPA axis disturbance is correlated with dietary habits and plasma fatty acid profiles. DESIGN: The participants were 41 women born during the first 6 months of 1960 and living in a Mediterranean area (Murcia, Spain). They were of normal weight, with a waist circumference of 80.5 +/- 9.3 cm. Their salivary cortisol levels, 7-day dietary record and plasma fatty acid profile were evaluated. Daily cortisol variability and postlunch cortisol secretion were recorded and a dexamethasone suppression test is performed in order to detect possible HPA disturbance. RESULTS: Both the methods used for HPA axis evaluation were positively correlated (r = 0.448, P = 0.004). Subjects with normal diurnal curves (high cortisol variability) showed significantly higher cortisol values in the morning and postprandial cortisol secretion than women with pathological curves (medium and low variability). Cortisol variability was inversely correlated with waist circumference (r = -0.312, P = 0.047), suggesting that a disturbed HPA axis response may lead to an android pattern of body fat distribution. Dietary fat and saturated fatty acid intake were lower in the high cortisol variability group, while monounsaturated fatty acid intake was higher (P < 0.05). No major differences were reported in plasma fatty acid profile. CONCLUSIONS: A disturbed HPA axis is associated with abdominal fat distribution and a higher content of fat and saturated fatty acids in the diet. Women who chose a dietary pattern closer to the Mediterranean diet, with high monounsaturated fatty acid intake, showed lower levels on HPA axis disturbance.

Relationship between fat cell size and number and fatty acid composition in adipose tissue from different fat depots in overweight/obese humans.

OBJECTIVE: To evaluate the body fat distribution and fat cell size and number in an overweight/obese population from both genders, and to determine the possible relationship between fat cell data from three different adipose tissue localizations (subcutaneous (SA), perivisceral and omental) and adipose tissue composition and dietary fatty acid. DESIGN: The sample consisted of 84 overweight/obese patients (29 men and 55 women) who have undergone abdominal surgery. The adipocyte size and total fat cell number was studied. Fat cell data were related with anthropometric, adipose tissue and subject's habitual diet fatty acid composition. MEASUREMENTS: Fat cell size was measured according to a Sjöström method from the three adipose depots. Total fat cell number was also calculated. The fatty acid composition of adipose tissue was examined by gas chromatography. The subjects diet was studied by a 7 days dietary record. RESULTS: Our data showed a negative relationship between the adipocyte size and the n-6 and n-3 fatty acids content of the SA adipose tissue (r=-0.286, P=0,040; r=-0.300, P=0.030) respectively, and the n-6 in the omental depots (r=-0.407, P=0.049) in the total population. Positive associations with the total of saturated (r=0.357, P=0.045) and negative (r=-0.544, P=0.001) with the n-9 fatty acids were observed when the relationship between the adipocyte number and the fatty acid composition of the different anatomical fat regions was studied. Dietary fatty acids composition positively correlated with fat cell size for the myristic acid (14:0) in men in the visceral depot (r=0.822, P=0.023), and for the saturated fatty acids (SFAs) in women in the omental depot (r=0.486, P=0.035). CONCLUSION: In the present study, for the first time in humans we found that n-3 and n-6 fatty acids are related to a reduced adipocyte size according to the depot localization. In contrast, adipose tissue and dietary SFAs significantly correlated with an increase in fat cell size and number. No significant associations were found between n-9 acids content and adipocyte size. However, n-9 adipose tissue fatty acids content was inversely associated with fat cell number showing that this type of fatty acid could limit hyperplasia in obese populations. The differences observed in the three different regions, perivisceral, omental and SA fat, indicate that this population adipose tissue have depot-specific differences.