India leads the world with largest number of diabetic subjects

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1 8 JAPI april 3 VOL. 6 Original Article Regulation of Adiponectin Secretion in Human and Adipose Tissue : Effects of Pioglitazone and Endothelin- : A Pilot Study Sujata R Mahadik, Ramchandra D Lele, Suresh D Mehtalia 3, Sudha S Deo 4, Vikram Parikh 5 Abstract Objective : The study was designed to test the effect of anti-diabetic agent pioglitazone and Endothelin- (ET-) on adiponectin secretion from human adipose tissue in depot dependent manner. Methods : adipose tissue (SAT) and omental adipose tissues (OAT) were obtained from 9 subjects, including 6 non-obese controls, 7 obese and 6 obese TDM patients. Adipose tissue was treated with pioglitazone and ET. Adiponectin secreted into the culture medium after treatment at different time interval (, 4, 48, 96 hours) was determined by ELISA and normalized for cellular DNA content. Results : Basal adiponectin secretion from both the depots significantly associated with serum adiponectin, BMI, waist and HOMA-IR. Though no depot-specific difference was found in adiponectin secretion from SAT and OAT in our population, significant reduction in adiponectin secretion was observed in SAT of obese and TDM patients compared to controls. Responsiveness to pioglitazone treatment was more in SAT, while ET inhibits adiponectin secretion in OAT. Conclusion : These data suggest that, SAT, appears to be major contributor to regulation of adiponectin in circulation. Pioglitazone stimulate adiponectin secretion in SAT compared to OAT in diabetic patients while ET- inhibiting adiponectin secretion in OAT of diabetic patients. We need to focus on mechanism underlying these regulatory agents mediated stimulation or inhibition of adiponectin secretion in human adipose tissue. Introduction India leads the world with largest number of diabetic subjects and will soon become the diabetes capital of the world. The main culprit of diabetes and related complications in Indians may be Asian Indian Phenotype which refers to higher waist circumference despite of lower body mass index (BMI) and increased prevalence of insulin resistance (IR)., The classical perception of adipose tissue as a storage place of fatty acids has been broadened over the last years. Recently it has been demonstrated that adipose tissue secrets various protein called adipokines (viz Leptin, Adiponectin, resistin, TNFa, IL-6), which influence insulin sensitivity profoundly, thereby providing a possible molecular link between increase adiposity and decrease insulin sensitivity of target tissue to insulin. -4 Of these adipokines, adiponectin has recently attracted much attention because of its efficient role against IR, obesity and TDM. Many studies in human proposed that plasma adiponectin levels are decreased in different metabolic disorder state like obesity and TDM and also negatively correlates with IR and obesity parameter. 5-8 The mechanism resulting in lowering the plasma adiponectin levels in obesity and TDM as opposed to other adipokines is not so clear. To gain insight into some of the regulatory processes affecting the plasma levels of adiponectin, it is necessary to compare adiponectin secretion in-vitro from major fat depots ie omental and subcutaneous. 9 In addition, adiponectin also exhibits novel insulin sensitizing and anti-atherogenic properties, which have made this adipokine Research Associate, Sir Hurkisondas Nurrotumdas Medical Research Society, Mumbai, India; Consultant Physician, Hon. Chief Physician and Director of Nuclear Medicine, Jaslok Hospital and Research Centre, Mumbai, India; 3 Diabetologist, 4 Senior Scientist, 5 Surgeon, Sir Hurkisondas Nurrotumdas Medical Research Society, Mumbai, India. Received: 3..; Revised: 7.6.; Accepted:.9. a promising, tool in the future for the treatment of TDM. Administration of adiponectin reverses IR in rodent models of obesity and TDM, but its direct administration might not be a good idea because of difficulties in maintaining high plasma levels. Thus the search for enhancer of endogenous adiponectin synthesis might be the most practical way for therapeutic application related to adiponectin. Hence in present study regulatory agents were selected to examine their effect on adiponectin secretion from subcutaneous and omental adipose tissue depots. First regulatory agent, Pioglitazone, the thiazolindinedione (TZD) class of anti-diabetic drug, is widely used for the treatment of diabetic patients with insulin resistance. The mechanism of pioglitazone to improve insulin sensitivity is not fully understood. In vivo and in vitro, TZD enhance the expression of adiponectin mrna and protein. -4 In present study we would like to confirm the effect of pioglitazone in stimulating adiponectin secretion from human adipose tissue and will also focus on its depot specific effect. Adipocytes are surrounded by stromal vascular cells, including endothelial cells, which secrete endothelin- (ET-) a potent vasoconstrictor. 5 Elevated levels of plasma ET- have been reported in various disease states including congestive heart failure, obesity and TDM. 6 ET- could promote systemic insulin resistance via direct or indirect effects on adipose tissue lipolysis. 7 Chronic ET- treatment leads to enhanced degradation of IRS-, decreased tyrosine phosphorylation of IRS- and G protein-q/ α-subunit in 3T3-L cells. There are findings indicating that ET- can directly and indirectly modulate adipocyte function. 7-9 Recently ET- has also been implicated as a regulatory agent in the secretion of adipose tissue derived adipokines like leptin, resistin and adiponectin from 3T3-L adipocytes. 8,9 Much more studies have concentrated on the role of ET- in glucose and lipid metabolism but there are no studies assessing effect of ET- on 44 JAPI april 3 VOL. 6

2 JAPI april 3 VOL. 6 9 Table : Characteristics of study subjects (Mean + SE) Parameter Control (n=6) Obese (n=7) TDM (n=6) M : F : 4 3 : 4 : 4 BMI (Kg/m ) Waist (cm) # # SBP (mmhg) DBP (mmhg) Glucose (mg/dl) # HOMA-IR Serum adiponectin level p <., p <., p <.5, # p <. for obese and TDM compared to controls. adiponectin secretion from human adipose tissue. Hence the second regulatory agent selected in the present study is ET-. To gain insight into metabolic role of adiponectin, this pilot study aims to evaluate differential regulation of adiponectin secretion from subcutaneous and omental adipose tissue depots by regulatory agents TZD and ET-. Materials and Methods Subjects The study population consisted of a total 9 subjects, consisting of 6 non-obese controls, 7 obese and 6 TDM patients. Diabetes was defined based on history (for patients taking oral hypoglycemic drugs) or according to WHO criteria of fasting glucose 7. mmol/l or hr glucose ³. mmol/l for subjects without a clinical history of diabetes. Most of the diabetic patients were receiving anti-diabetic agent sulphonylurea, metformin or combination of both. Obesity was defined if their BMI 5 kg/ m according to cut-off suggested for Asian Indians. Controls were classified as having normal glucose tolerance (Fasting plasma glucose < 6. mmol/l and hrs glucose < 7.8 mmol/l). These subjects were surgical patients from H. N. Hospital and St George s Hospital, Mumbai, who underwent abdominal surgery. All subjects were fasted overnight before tissue removal. adipose tissue (SAT) and omental adipose tissues (OAT) (~ -5g) were collected during the surgical procedure and were immediately sent to the laboratory. SAT was collected at the site of transverse lower abdominal incision and OAT was collected from greater omentum. All the subjects gave their informed consent after the procedure was explained to them. The ethics committee of Sir Hurkisondas Nurrotumdas Hospital and Medical Research Society approved the project. The subjects had an age range of 3-7 years. Anthropometric measurement including height, weight and waist circumference (abdominal circumference at the level of iliac crest) and clinical details including blood pressure measurement were obtained from the case report. BMI was calculated from the ratio of body weight in kg to height in square meters and expressed as kg/m units. Fasting blood samples were also obtained after surgery for the determination of serum adiponectin, insulin and plasma glucose levels. Insulin resistance measured as Homeostasis Model Assessment-Insulin Resistance (HOMA-IR) using following formula : Fasting insulin (µu/ml) x Fasting glucose (mmol/l)/. Materials General reagents were of the highest available grade and obtained from Sigma (St.Louis, MO). DMEM:Ham F culture medium, Endothelin were from Sigma (St.Louis, MO), HBSS Buffer from(invitrogen, USA). Pioglitazone was generously provided by Franco-Indian (Mumbai, India). Fasting plasma glucose was measured by the glucose peroxidase method (Randox, San Francisco, Calif., USA). Elisa Kits for human serum adiponectin, insulin were obtained by linco Res Inc (St.Charls, MO, USA). DNA was extracted using the Hipur mammalian genomic DNA extraction kit (HiMedia, Mumbai, India). Adipose Tissue Organ Culture Adipose tissue contains many cell types, including adipocytes, endothelial cells, pre-adipocytes, and fibroblasts. Thus, organ culture preserves paracrine interactions among cells that can influence adipocyte metabolism. The major strength of this method is the good maintenance of gene expression and adipocyte function within adipose tissue placed in organ culture for up to weeks. The subcutaneous and omental adipose tissue collected during abdominal surgery is processed under sterile conditions. The tissue was washed several times with HBSS, to remove the majority of connective tissue and blood clots. The adipose tissue (~ mg) was cut into fragments and then placed into 35mm Petri dishes with.5ml of DMEM:Ham F culture medium without Fetal Bovine Serum (FBS) incubated under an atmosphere of 5% CO at 37 o C. 5 The adipose tissue was preincubated for 4 hours, and then the medium was replaced with fresh medium containing the indicated substances. The incubation continued for the time indicated (maximum 96 hour). Pioglitazone was added to achieve a final concentration of μm. Endothelin was added to achieve a final concentration of nm. The above concentration of regulatory agents was chosen, since these concentrations are suggested to elicit maximal biological effects as found in adipose tissue 4 or other in-vitro cell system. 5 Samples of conditioned medium were taken every, 4, 48 and 96 hours and immediately frozen and maintained at -8 o C until use. Medium with un-treated cells was used as a blank control (Basal level). Measurement of Adiponectin in Culture Medium ELISA was used to measure the secretion of adiponectin into the medium of the cultured human adipose tissue explants. According to the kit manufacturer s directions (Linco Research, Inc.) samples of culture medium were analyzed and human adiponectin standard provided in the kit was used as standard. To normalize for cell number in the assay, DNA was isolated from aliquots of adipose tissue explants and quantified using spectrophotometer. Adiponectin secretion was reported as nanogram per ml of culture medium per microgram of cell DNA. 9 Statistical Analysis Quantitative data is expressed as Mean ± SE. Group means were compared using one way ANOVA or unpaired t test. To determine the relationship between adiponectin secretion and different parameters of metabolic syndrome, a Bi-variate correlation analysis was used. A p value of less than.5 was considered as statistically significant. All analysis was performed using SPSS (Version 6). Results The clinical characteristics of study subjects including 6 non-obese controls, 7 obese and 6 TDM patients are shown in Table. Mean levels of BMI, waist size, plasma glucose and HOMA-IR were significantly increased and serum adiponectin levels significantly reduced in obese subjects compared to control subjects. In case of diabetic patient mean levels of BMI, waist size, systolic and diastolic blood pressure, plasma glucose were significantly increased and serum adiponectin levels significantly reduced compared to control subjects. No difference was found in HOMA-IR levels may be due to less number of patients or JAPI april 3 VOL. 6 45

3 JAPI april 3 VOL. 6 Adiponectin Secretion (ng/ml per Hg Non Obese Control Fig. (a, b, c) : Presents adiponectin secretion (ng/ml per μg from SAT and OAT of non-obese control, obese and TDM subjects at different time intervals (,4,48,96 hours). Results were presented as Mean ± SE due to effect of anti-diabetic treatment. Interestingly, In Pearson correlation analysis in all subjects, basal adiponectin secretion (expressed as ng/ml per DNA content) from SAT is significantly correlated with serum adiponectin (r =.84, p<.), BMI (r = -.543, p<.5) and waist size (r = -.46, p<.5). It also exhibited negative association with HOMA-IR, but not able to reach statistical significance (r =-.44). In case of OAT, adiponectin secretion is significantly correlated with serum adiponectin (r =.86, p<.), BMI (r = -.575, p<.5) and waist size (r = -.4, p<.5). It also exhibits significant association with HOMA-IR (r = -.76, p<.5). Basal level adiponectin secretion The time course study was performed for up to 96 hours and a Time in hours c b Obese subjects Time in hours TDM Patients adiponectin secretion expressed as ng/ml per DNA content. In time course study adiponectin secretion increased with increasing time (from, 4, 48, 96 hrs.) in control, obese and diabetic subjects. Depot-specific comparison No depot-specific difference was found in adiponectin secretion from SAT and OAT in control, obese and diabetic subjects [Figure (a, b, c)]. Metabolic disease condition based comparison: Comparison of adiponectin secretion of obese and diabetic patients with non obese control from SAT was demonstrated in Figure a, where adiponectin secretion significantly reduced in obese and TDM patients compared to control in SAT (p <.5). Figure b presents comparison of adiponectin secretion of obese and diabetic patients with non-obese control from OAT. Where, adiponectin secretion was reduced in obese and TDM patients compared to control but not able to reach statistical significance in OAT. Effect of regulatory agent on adiponectin secretion: Depot-specific comparison The effect of two regulatory agents, pioglitazone and endothelin- on adiponectin secretion were observed in present study and results were expressed as percentage (%) increase or decrease in adiponectin secretion at 96 hr after treatment with regulatory agent compared to untreated cells. When the adipose tissues were treated with pioglitazone, adiponectin secretion was stimulated from both the depots. Though stimulation of adiponectin secretion was higher in SAT than OAT, statistically significant effect was observed only in TDM patients (Table ). Interestingly inhibitory action of ET- on adiponectin secretion was found from both the depots. Statistically significant inhibition of adiponectin secretion was found in OAT compared to SAT depot of diabetic patients only (Table ). Metabolic disease condition based comparison In present study the effect of Pioglitazone and ET- on adiponectin secretion on the basis of metabolic disease condition exhibited no difference in adiponectin secretion from SAT of both obese and diabetic patients compared to control subjects. In OAT, no difference in adiponectin secretion was found as effect of pioglitazone but interestingly, ET- shows significant decrease in adiponectin secretion of diabetic patient compared to control. ( vs , p <.5) (negative value presents the % decrease in adiponectin secretion). Discussion To gain insight into metabolic role of adiponectin, in this pilot study we demonstrated differential regulation of adiponectin secretion from subcutaneous and omental adipose tissue depots. The effect of regulatory agents TZD and ET- on adiponectin secretion was also examined. Importance of adipose tissue secretion of adiponectin for regulating amount of circulating adiponectin was reported in few studies. Findings from these studies were inconsistent, since few studies reported that adiponectin release from omental adipocyte accounts for the reduced serum adiponectin observed in obesity. 9,3 While others found that SAT is important for regulation of adiponectin levels than OAT.,4,4-7 This could be due to visceral (omental) adipose tissue is only small fraction of total adipose tissue, even in obese subjects, as stated by the previous literature. 8 In the present study we demonstrated that adiponectin 46 JAPI april 3 VOL. 6

4 JAPI april 3 VOL. 6 Adiponectin Secretion (ng/ml per μg.5.5 Control Obese TDM a SAT Time in Hours Fig. (a, b): Presents comparison of adiponectin secretion (ng/ml per μg from SAT and OAT of obese and TDM patients with non-obese control subjects at different time intervals (,4,48,96 hours). Results were presented as Mean ± SE. p <.5 for obese and TDM compared to non-obese controls. Table : Effect of Pioglitazone and ET- on Adiponectin secretion from SAT compared to OAT at different metabolic states Control Obese TDM Pioglitazone SAT OAT n=6 Endothelin- SAT n= OAT n=7 secretion from SAT is significantly lowered in TDM compared to non-obese control subjects. Thus may be contributing to decreased serum adiponectin levels. In OAT, the adiponectin secretion was decreased in both the metabolic disorder condition (obesity, TDM) but not able to reach statistical significance may be due to wide distribution of adiponectin secretion among unrelated human subjects. Interestingly, reduction in adiponectin secretion in obese and TDM compared to non-obese control subjects was found more in SAT (approx 5-6 % reduction) than in OAT (approx 3-4 % reduction). To better understand the impact of regional fat distribution on adiponectin secretion, comparison of adiponectin secretion from SAT and OAT of individual control obese and diabetic patients were carried out. Depots differences in adiponectin gene expression and secretion have been reported by some but not all investigators and importantly are not always correlated with circulating levels. 5,7,9,3 In the present study no depot specific effect in adiponectin secretion was found at basal level irrespective of underlying metabolic disease conditions like non-obese control, obese and TDM. These findings are consistent with previous reports on depot dependent study of adiponectin secretion from cultured adipocyte or adipose tissue. 9,3,3,5 In addition to depot specific effect at basal level, few studies have also demonstrated that adiponectin expression and secretion from adipose tissue are regulated by a wide variety of regulatory agent including insulin, TZDs, TNF α, β adrenergic agonists, IL6 etc. 9,3-33 In the present study, the responsiveness of adiponectin secretion to the treatment of antidiabetic agent pioglitazone and vasoconstrictor endothelin- was determined. The effect of regulatory agent at both depot specific manners as well as at different metabolic condition was examined p <.5 for SAT compared to OAT. Results were expressed as percentage increase or decrease in adiponectin secretion at 96 hr after treatment with regulatory agent compared to untreated cells. (Negative value indicates the % decrease in adiponectin secretion) Control Obese TDM b OAT Time in Hours In the present study pioglitazone significantly increased adiponectin secretion from cultured SAT explants as compared to OAT mainly obtained from diabetic patients. These findings are consistent with in vivo and in vitro studies -4 where they reported TZD s enhance the expression of adiponectin mrna and protein from SAT. Though Motoshima et al reported contradictory findings that TZD augment adiponectin secretion from OAT but not SAT. 9 Thus response revealed to TZD treatment by SAT in current study might be contributing to increased adiponectin levels in circulation after TZD treatment. We need to focus on mechanism underlying TZD mediated stimulation of adiponectin secretion in human adipose tissue. Recently ET- has also been implicated as a regulatory agent in the secretion of adipocyte derived secretory factor like leptin, resistin and adiponectin from 3T3L adipocyte. These studies in 3T3L adipocyte demonstrated that ET- inhibits basal and insulin stimulated adiponectin secretion through phosphatidylinositol 4, 5 bisphosphate (PIP) modulation of actin cytoskeleton. 8,9 But virtually, no data is available where effect of ET- on adiponectin secretion from human adipose tissue was studied. For the first time we report that effect of ET- inhibits adiponectin secretion in omental adipose tissue of TDM patients compared to non-obese control subjects. We need to focus on the mechanism of action responsible for the effects of ET- on adiponectin secretion in omental fat of diabetic patients. Limitation of this study is the limited number of sample in each group. Since it is very difficult to obtain subcutaneous and omental adipose tissue samples at different metabolic condition from surgically operated subjects. Conclusion In summary, this pilot study demonstrated that, in diabetic condition decreased adiponectin secretion in SAT may be contributing to regulation of adiponectin in circulation. Further studies are needed with large number of patients to confirm this finding. Interestingly depot specific studies on effect of regulatory agents reveal that pioglitazone stimulates adiponectin secretion in SAT compared to OAT in diabetic patients while ET- inhibits adiponectin secretion in OAT of diabetic patients. We also need to focus on mechanism underlying different regulatory agent mediated stimulation or inhibition of adiponectin secretion in human adipose tissue. Acknowledgement The Sir H.N. Medical Research Society supported this work. We thank Mr. Parag and Ms Deepika for their kind assistance in this project. We also thank Dr. S Borude and Dr. M Naik for their kind support. Conflict of Interest There is no conflict of interest. JAPI april 3 VOL. 6 47

5 JAPI april 3 VOL. 6 References. Misra A, Vikram NK. Insulin Resistance Syndrome and Asian Indian. Current Science ;83: Mohan V, Deepa R. Adipocytokine and Expanding Asian Indian Phenotype. JAPI 6;54: Fasshauer M, Paschke R, Stumvoll M., Adiponectin, obesity, and cardiovascular disease. Biochimie 4;86: Kadowaki T, Yamauchi T, Kubota N, Hara K, Veki K, Tobe K. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J Clin Investigation 6;6: Bruun JM, Lihn AS, Verdich C, Pedersen SB, Toubro S, Astrup A, Richelsen B. Regulation of adiponectin by adipose tissue derived cytokines : in vivo and in vitro investigations in humans. Am J Physiol Endocrinol Metab 3;85:E57-E Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y et al. Plasma concentration of a novel, adipose specific protein adiponectin, in Type diabetic patients. Arterioscler Thromb Vasc Biol ;: Arita Y, Kihara S, Ouchi N, Matsuzawa Y. Paradoxical decrease of an adipose specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 999;57: Statnick MA, Beavers LS, Conner LJ, Caro JF. Decreased expression of apmi in omental and subcutaneous adipose tissue of humans with type diabetes. Int J Exp Diabetes Res ;: Motoshima H, Wu X, Sinha MK Goldsetin BJ et al. differential regulation of Adiponectin Secretion from cultured human and Adipocytes : Effect of insulin and Rosiglitazone. J Clin Endocrinol Metab ;87: Han SH, Quon MJ, Kim J, Koh KK. Adiponectin and Cardiovascular Disease, response to therapeutic Intervention. J Am Coll Cardiol 7;49: Rasouli N, Yao-Borengasser Y, Miles LM, Elbein SC, Kern PA. Increased plasma adiponectin in response to pioglitazone does not result from increased gene expression. Am J Physiol Endocrinol Metab 6;9:E4-E46.. Maeda N, Takahashi M, Funahashi T, Kihara S, Nishizawa H, Kishida K et al. PPAR gamma ligands increase expression and plasma concentrations of adiponectin, an adipose derived protein. Diabetes ;5: Philips SA, Ciaraldi TP, Kong AP, Bandukwala R, Aroda V, Carter L et al. Modulation of circulating and adipose tissue adiponectin levels by anti-diabetic therapy. Diabetes 3;5: Philips SA, Ciaraldi TP, Oh DK, Savu MK, Henry RR. Adiponectin secretion and response to pioglitazone is depot dependent in cultured human adipose tissue. Am J Physiol Endocrinol Metab 8;95:E84-E Clarke KJ, Zhong Q, Schwartz DD, Coleman ES, Kemppainen RJ, Judd RL. Regulation of adiponectin secretion by endothelin-. Biochemical and Biophysical Research Communications 3;3: Decker ER, Brock, TA in Endothelin: Molecular Biology, Physiology, and Pathology (Highsmith, RF, ed) 998;93 9, Humana Press Inc., Totowa, NJ. 7. VanHarmelen V, Eriksson A, Astrom G, Wahlen K, Naslund E, Karpe F et al. Vascular Peptide Endothelin- Links Fat Accumulation with Alterations of Visceral Adipocyte Lipolysis. Diabetes 8;57: Y. Xiong, H. Tanaka, J. A. Richardson, S. C. Williams, C. A. Slaughter, M. Nakamura, etal. Endothelin- Stimulates Leptin Production in Adipocytes. J Biol Chem ;76: Zhong Q, Lin CY, Clarke KJ, Kemppainen RJ, Schwartz DD,Judd RL. Endothelin- inhibits resistin secretion in 3T3-Ladipocytes. Biochem Biophys Res Commun ;96: World Health Organization. Definition, diagnosis and classifiation of Diabetes Mellitus and its complications. Report of a WHO consultation. Part : Diagnosis and classification of Diabetes Mellitus (WHO /NCD / CS / 99.) World Health Organization, Geneva (999).. World Health Organization. The Asia-Pacific perspective: Redefining obesity and its treatment. Geneva, Switzerland: World Health Organization.. Matthews, D.R., Husker, J.P., Rudenski, A.S., Naylor, B.A., Treacher, D.F. and Turner R.L., Homeostasis model assessment: Insulin resistance and Beta Cell Function from fasting plasma glucose and insulin concentrations in man. Diabetologia 985;8: Drolet R, Belanger C, Fortier M, Huot C, Mailloux J. Fat depot specific impact of visceral obesity on adipocyte adiponectin release in womens. Obesity 8;7: Lihn AS, Bruun JM, He G, Pedersen SB, Jensen PF, Richelsen B. Lower expression of adiponectin mrna in visceral adipose tissue in lean and obese subjects. Mol cell endocrinol 4;9: Degawa-Yamauchi M, Moss KA, Bovenkerk JE, Shankar SS, Morrison CL, Considine RV etal. Regulation of adiponectin expression in human adipocytes: Effect of Adiposity, Glucocorticoids and Tumor Necrosis Factor alpha. Obesity Res 5;3: Fain JN, Madan A K, Hiler ML, Cheema P, Bahouth SW. Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocyte from visceral and subcutaneous abdominal adipose tissue of obese humans. Endocrinology 4;45: Fisher FM, Mcternan PG, Valsamakis G, Chetty R, Harte AL et al. Differences in adiponectin protein expression: effect of fat depots and type II diabetes status. Horm Metab Res ;34: Ross R, Shaw KD, Martel Y, de Guise J, Avruch L. Adipose tissue distribution measured by magnetic resonance imaging in obese women. Am J Clin Nutri 993;57: Krotkiewski, M., Bjorntorp, P., Sjostrom, L, Smith U. Impact of obesity on metabolism in men and women, Importance of regional adipose tissue distribution. J Clin Invest 983;7: Perrini S, Laviola L, Cignarreli A, Melchiorre M, De Stefano F, Caccioppolic C, Natalicchio A et al. Fat depot- related differences in gene expression, adiponectin secretion and insulin action and signaling in human adipocytes differentiated in vitro from precursor stromal cells. Diabetologia 8;5: Fasshauer M, Paschke R. Regulation of adipocytokines and insulin resistance, Diabetologia 3;46: Kappes A., Loffler G. Influences on ionomycin, di-butyryl-cyclo AMP and Tumor Necrosis Factor alpha on intracellular amount and secretion of apm in differentiating primary human pre-adipocytes. Horm Metab Res ;3: Halleux CM, Takahashi M, Delporte ML, Detry R, Funahashi T, Matsuzawa Y e al. Secretion of Adiponectin and Regulation of apm gene expression in Human Visceral Adipose tissue, Biochem Biophys Res Commun ;88: JAPI april 3 VOL. 6