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Nature

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01/19/2013

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Original Article
Obesity Research
(2005)
13
, 662
 – 
669; doi: 10.1038/oby.2005.74
Regulation of Adiponectin Expression in HumanAdipocytes: Effects of Adiposity, Glucocorticoids, andTumor Necrosis Factor 
Mikako Degawa-Yamauchi
*
,Katherine A. Moss
*
,Jason E. Bovenkerk 
*
,Sudha S. Shankar
*
, Charles L. Morrison
,Christopher J. Lelliott
,Antonio Vidal-Puig
,RoseMarie Jones
 andRobert V. Considine
*
 1.
 
*
Division of Endocrinology and Metabolism, Department of Medicine, IndianaUniversity School of Medicine; Indianapolis, Indiana2.
 
Department of Surgery, Indiana University School of Medicine; Indianapolis, Indiana3.
 
St. Vincent Bariatric Services, Carmel, Indiana4.
 
§
Department of Clinical Biochemistry, Addenbrook's Hospital, Cambridge, UnitedKingdomCorrespondence: Robert V. Considine, Indiana University School of Medicine, 541 NorthClinical Drive, Clinical Building 455, Indianapolis, IN 46202-5111. E-mail: rconsidi@iupui.edu 
**
The costs of publication of this article were defrayed, in part, by the payment of page charges.This article must, therefore, be hereby marked "advertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.Received 9 July 2004; Accepted 14 January 2005.Top of page 
Abstract
Objective:
Adiponectin mRNA expression in isolated subcutaneous and omental adipocytes wasexamined across a wide range of adiposity to determine whether adiponectin synthesis isimpaired in these adipose tissue depots in obese humans. Tumor necrosis factor (TNF) anddexamethasone were tested for inhibitory effects on adiponectin release from human adipocytesin vitro.
 Research Methods and Procedures:
Adipocytes were isolated by collagenase digestion of abdominal adipose tissue obtained from subjects undergoing surgical procedures or outpatientneedle biopsy. Adiponectin and leptin mRNA were quantitated by real-time reverse
 
transcriptase-polymerase chain reaction. Adiponectin and leptin secretion from isolatedadipocytes treated with dexamethasone or TNF were determined by radioimmunoassay.
 Results:
There was a significant negative correlation between adiponectin gene expression andBMI in subcutaneous adipocytes from 32 women (
= 0.420;
 p
= 0.02). Adiponectin mRNA wasalso significantly correlated with serum adiponectin (
= 0.44;
 p
= 0.03;
n
= 25). There was nocorrelation between adiponectin mRNA expression and BMI in omental adipocytes from 29women. Leptin mRNA was significantly and positively correlated (
= 0.484;
 p
= 0.01) withBMI in the same omental adipocyte mRNA preparations. In subcutaneous adipocytes from leansubjects, TNF inhibited adiponectin release by 7.4 1.2% (
n
= 9,
 p
< 0.05) but had no effect onadiponectin release from subcutaneous or omental adipocytes from obese subjects.Dexamethasone significantly inhibited adiponectin release with 24 hours of treatment.
 Discussion:
The data suggest that reduced adiponectin synthesis in subcutaneous adipocytescontributes to lower serum adiponectin levels in obesity and that glucocorticoids regulateadiponectin gene expression in human adipocytes. TNF does not seem to directly inhibitadiponectin synthesis in human adipocytes.
Keywords:
adiponectin, tumor necrosis factor , human adipocytesTop of page 
Introduction
Adipose tissue is an endocrine organ producing many hormones and cytokines(1,2,3,4). The serum concentration of adipose tissue secretory products is generally increased in obese humansbecause of the combination of a greater number of adipocytes and, for some factors such asleptin, increased secretion from the larger adipocytes present in obese individuals(5). Elevated serum levels of adipose tissue secretory products have been linked to the development of insulinresistance, diabetes, and cardiovascular disease. In contrast, serum levels of the adipose tissuehormone adiponectin are significantly reduced in obese adults(6,7,8)and children(9,10), and this reduction seems to be linked to subsequent development of insulin resistance and diabetes(11,12). Adiponectin is a collagen-like molecule(13,14,15), and observations in rodents and cultured cell models show that this novel protein has antiinflammatory, antiatherogenic, and insulin-sensitizing properties. Adiponectin suppresses tumor necrosis factor (TNF)
1
 production fromcultured macrophages(16)and inhibits TNF-induced monocyte attachment to endothelial cells through a reduction in adhesion molecule expression(17). Adiponectin blocks the binding of  oxidized low-density lipoproteins to macrophages and reduces their cholesterol content(18). Adiponectin inhibits growth factor
 – 
induced proliferation of smooth muscle cells(19)and protects against intimal thickening in injured vessels(20). Administration of physiological concentrations of adiponectin to lipoatrophic mice lacking the hormone reduces liver and muscletriglyceride content and reverses insulin resistance(21). The hormone also improved insulin
 
sensitivity in genetically obese and diet-induced obese mice(21). The globular domain of  adiponectin induced weight loss in mice through increased fatty acid oxidation(22)and has also been shown to increase glucose uptake in isolated rat adipocytes(23). It can thus be concluded from animal and in vitro work that the lower adiponectin levels observed in obese humans likelycontribute to insulin resistance/diabetes and vascular disease.The mechanism(s) resulting in lower serum adiponectin in obese humans is not known. Areduction in adiponectin gene expression and secretion in adipose tissue from obese and obesediabetic subjects has been found in some(13,24,25,26,27), but not all(28), studies. The reason for this discrepancy is not clear. Furthermore, the role of omental vs. subcutaneous adipose tissueproduction of adiponectin has not been fully addressed. Therefore, in this study, we examinedthe expression of adiponectin mRNA in human subcutaneous and omental adipocytes obtainedfrom women across a wide range of adiposity. In addition, we tested the hypothesis, based onobservations in 3T3-L1 cells(29), that TNF and glucocorticoids are significant inhibitors of  adiponectin synthesis and release from human adipocytes.Top of page 
Research Methods and Procedures
Adipose Tissue Biopsy and Cell Culture
Subcutaneous and omental adipose tissue biopsies were obtained from subjects undergoingbariatric surgical procedures. Subcutaneous adipose tissue from lean and obese subjects was alsoobtained by outpatient needle biopsy(30). Omental adipose tissue samples were also obtained from three lean women during open abdominal surgery for ulcerative colitis, stomach cancer,and colostomy. No subjects were taking diabetic medications (thiazolidinediones, insulin) thatmight affect adiponectin expression. All subjects provided informed consent, and theInstitutional Review Boards of Indiana University-Purdue University at Indianapolis, IN, and St.Vincent's Hospital, Indianapolis, IN, approved all protocols.Adipocytes were isolated by collagenase (1 mg/mL) digestion as previously described(31)and used directly for RNA isolation or placed into culture. Adipocytes (1 mL packed cells) weresuspension cultured in 5 mL Dulbecco's modified eagle medium (DMEM)/nutrient mixture F12+ 10% fetal bovine serum in 50 mL polypropylene centrifuge tubes kept on their sides at a 15°angle above horizontal, 37 °C, and 5%CO
2
. The culture medium was replaced with freshmedium every 24 hours. Collagenase was from Worthington Biochemicals (Freehold, NJ).Culture medium (DMEM; D5523 and Nutrient Mixture F12; N6760) and dexamethasone(D2915; water soluble) were purchased from Sigma Chemical (St. Louis, MO). Fetal bovineserum was from Life Technologies (Grand Island, NY). TNF (210TA) was from R&D Systems(Minneapolis, MN).3T3-L1 adipocytes were differentiated to mature adipocytes by standard techniques(32)and were used between 7 and 14 days after initiation of differentiation.
Assays

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