The purpose of this study was to examine the source of adipokines released by the visceral and sc adipose tissues of obese humans. Human adipose tissue incubated in primary culture for 48 h released more prostaglandin E(2), IL-8, and IL-6 than adiponectin, whereas the release of plasminogen activator inhibitor 1 and hepatocyte growth factor was less than that of adiponectin but greater than that of leptin. IL-10 and TNFalpha were released in amounts less than those of leptin, whereas vascular endothelial growth factor and IL1-beta were released in much lower amounts. The accumulation of adipokines was also examined in the three fractions (adipose tissue matrix, isolated stromovascular cells, and adipocytes) obtained by collagenase digestion of adipose tissue. Over 90% of the adipokine release by adipose tissue, except for adiponectin and leptin, could be attributed to nonfat cells. Visceral adipose tissue released greater amounts of vascular endothelial growth factor, IL-6, and plasminogen activator inhibitor 1 compared with abdominal sc tissue. The greatly enhanced total release of TNFalpha, IL-8, and IL-10 by adipose tissue from individuals with a body mass index of 45 compared with 32 was due to nonfat cells. Furthermore, most of the adipokine release by the nonfat cells of adipose tissue was due to cells retained in the tissue matrix after collagenase digestion.

No abstract

OBJECTIVE:The primary aim was to investigate the relative importance of the adipocytes vs the nonfat cells present in human adipose tissue with respect to release of immunoreactive tumor necrosis factor-a (TNFa). The second aim was to examine the correlation between body mass index (BMI) and the subsequent release of adiponectin and TNFa by explants of human subcutaneous and visceral adipose tissue incubated in primary culture for 48 h. RESULTS: We found that the maximal release of TNFa was seen during the first 4 h of a 48-h incubation by explants of human adipose tissue in primary culture. Over 95% of the TNFa released to the medium by human adipose tissue explants over a 4-h incubation came from the nonfat cells present in the adipose tissue. The release of TNFa by the nonfat cells released during collagenase digestion was slightly higher than that by the cells present in the adipose tissue matrix after collagenase digestion. TNFa release by the combined matrix and isolated nonfat cells was greater than that by explants of tissue indicating some upregulation induced by collagenase digestion. Immunoreactive TNFa disappeared from the medium with a half-time of approximately 10 h. There was a positive correlation coefficient of 0.79 between TNFa release by tissue explants and the BMI of the fat donors as well as a correlation of 0.52 between BMI and release by adipocytes. TNFa release negatively correlated [À0.60] with adiponectin release by adipose tissue. The release of TNFa was far less than that of adiponectin or IL-6, and less than that of plasminogen activator inhibitor-1, hepatocyte growth factor, or leptin over a 4-h incubation of human adipose tissue explants. TNFa release over 4 h was enhanced by lipopolysaccharide and inhibited by a cyclooxygenase-2 inhibitor. CONCLUSION: The release of TNFa by adipose tissue of obese humans is primarily due to the nonfat cells present in adipose tissue. TNFa is a short-lived adipokine whose release by human adipose tissue in primary culture correlates with the BMI of the fat donors.

Because UCP-1 is expressed at high levels in epicardial fat as compared to other fat depots, the possibility should be considered that epicardial fat functions like brown fat to defend the myocardium and coronary vessels against hypothermia. This process could be blunted in the elderly.

The sympathetic nervous system mediates its regulatory effects through G protein-coupled receptors (GPCR) 3 related to the family of ␣-and ␤-adrenergic receptors. Among these receptors is the ␤ 1 -AR, which is coupled to the G s -cyclic AMP axis and plays a major role in transmitting sympathetic regulation to cardiac, renal, vascular, and other organs (2, 3). Persistent activation of the ␤ 1 -AR or other GPCR causes their desensitization and internalization via clathrin-coated pits or caveolae into early endosomes (4 -6). Internalized GPCR are either recycled back to the cell surface for another round of signaling or retained for degradation by lysosomal or proteasomal pathways (7-9). Characterization of the players involved in these distinct outcomes is the purpose of this study.Recycling and resensitization of the ␤ 1 -AR are dependent upon two motifs; one is the ESKV sequence in the carboxylterminal tail, and the other is the region surrounding Ser 312 in the 3rd IC of the ␤ 1 -AR (10, 11). The ESKV tetrapeptide conforms to a type I (PSD-95/DLG/ZO1) PDZ ligand (i.e. X(S/ T)X⌽, where X at positions Ϫ1 and Ϫ3 is any amino acid, and ⌽ at position 0 is a hydrophobic amino acid) (12, 13). Mutagenesis of the type 1 PDZ or Ser 312 to alanine prevented the recycling and resensitization of the ␤ 1 -AR (10, 11). Concerning Ser 312 , we determined that this residue is specifically phosphorylated by PKA and that the activity of PKA was required for recycling and resensitization of the human ␤ 1 -AR (11).These results indicate that two distinct motifs are involved in recycling of the ␤ 1 -AR, but they do not explain how they crosstalk to one another to coordinate the sequence of events involved in recycling of this GPCR. A major breakthrough in identifying the mechanism of cross-talk between these two motifs was the identification of AKAP79 as the AKAP involved in recycling of the ␤ 1 -AR in HEK-293 and other cell lines (1). AKAP79 promoted the targeting of PKA to the ␤ 1 -AR by binding to the carboxyl-terminal 53 amino acids (between residues 425 and 477) of the ␤ 1 -AR (1). Here we report that the binding domain of AKAP79 to the ␤ 1 -AR overlaps with its type 1 PDZ motif. However, the binding between the PDZ and AKAP79 is indirect and involves the MAGUK protein SAP97 that simultaneously binds to AKAP79 and type 1 PDZ to target PKA to the * This work was supported by Grant-in-aid 6071785 from the Southeastern Affiliate of the American Heart Association (to S. W. B.) and by an NIDDK grant from the National Institutes of Health (to A. P. N.). 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.

UCP-1 is relatively abundant in epicardial fat, and this depot possesses molecular features characteristic of those found in vitro in beige lineage adipocytes.

PDE3A functionally and physically interacts with CFTR. Inhibition of PDE3A generates compartmentalized cAMP, which further clusters PDE3A and CFTR into microdomains at the plasma membrane of epithelial cells and potentiates CFTR channel function. Our findings provide insights into the important role of PDE3A in compartmentalized cAMP signaling.

Resensitization of G protein-coupled receptors (GPCR)folThe ␤ 1 -AR 2 is a major receptor for the physiological regulation of cardiac function by the sympathetic nervous system and plays an important role in clinical management of hypertension and heart failure (2). Agonist-mediated activation of the ␤ 1 -AR results in the generation of intracellular cyclic AMP and in the activation of PKA, which in turn phosphorylates numerous intracellular targets that mediate the familiar effects of ␤-agonists (3). As a consequence of their activation, the ␤ 1 -AR and other G protein-coupled receptors (GPCR) can undergo desensitization, which is characterized by attenuation of GPCR signaling intensity (4). The biochemical mechanisms of desensitization are numerous, but appear to be initiated by the phosphorylation of the agonist-occupied GPCR by G protein-coupled receptor kinases (GRK), followed by uncoupling of the GPCR from its cognate G protein by ␤-arrestins, which culminates in the internalization or sequestration of the GPCR away from its signaling platform (5, 6). Internalization of the GPCR appears to produce different outcomes depending on the type of GPCR and cell line under study. For ␤ 1 -AR, ␤ 2 -AR, and other GPCR, internalization is a prerequisite for resensitization because intracellular trafficking and subsequent recycling of resensitized GPCR promotes their insertion into the cell membrane to maintain agonist responsiveness (1,(7)(8)(9). For the ␦-opioid, other GPCRs, the internalized receptors do not recycle back, instead they are retained intracellularly and later degraded either by lysosomal or proteasomal pathways (10 -12).Recently, PKA and its putative substrate, serine at position 312 (Ser 312 ) in the third intracellular loop (3 rd IC) of the human ␤ 1 -AR, were found to be critical determinants of the ability of the ␤ 1 -AR to recycle and resensitize (1). Inhibition of PKA or mutagenesis of Ser 312 to alanine (S312A) prevented the recycling and resensitization of the agonist-internalized ␤ 1 -AR (1). PKA-mediated resensitization of the ␤ 1 -AR is termed "homologous resensitization" because PKA is activated through the * This work was supported by a grant-in-aid from the Southeastern affiliate of the American Heart Association and National Institutes of Health Grants HL-71419 (to S. W. B.), NS-46661 (to S. J. T.), and GM-48231 (to J. D. S.). 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.