Suleiman W. Bahouth

Resistin release by human adipose tissue explants in primary culture

Resistin, also known as Fizz3 or ADSF, is a protein found in murine adipose tissue and inflammatory lung exudates. The present studies found that resistin was released by explants of human adipose tissue but the release was quite variable ranging from 3 to 158 ng/g over 48 h. The release of resistin was 250% greater by explants of omental than by explants of human subcutaneous abdominal adipose tissue. Resistin release by adipocytes was negligible as compared to that by the non-fat cells of adipose tissue. Leptin formation by adipocytes was 8-fold greater than its formation by the non-fat cells, while the formation of PAI-1 by adipocytes was 38% of that by the non-fat cells. The conversion of glucose to lactate as well as the formation of PGE(2) and IL-8 was approximately 15% of that by the non-fat cells. In contrast the release of IL-6 and IL-1beta by adipocytes was 4-7% of that by the non-fat cells while the formation of resistin and IL-10 by adipocytes was 2% of that by non-fat cells. The release of adiponectin by explants ranged from 1000 to 5000 ng/g over 48 h but did not correlate with that of resistin. The present data suggest that resistin release by explants of human adipose tissue in primary culture is largely derived from the non-fat cells present in the explants.

TNFα release by the nonfat cells of human adipose tissue

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-α (TNFα). The second aim was to examine the correlation between body mass index (BMI) and the subsequent release of adiponectin and TNFα by explants of human subcutaneous and visceral adipose tissue incubated in primary culture for 48 h.RESULTS: We found that the maximal release of TNFα 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 TNFα 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 TNFα 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. TNFα 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 TNFα disappeared from the medium with a half-time of approximately 10 h. There was a positive correlation coefficient of 0.79 between TNFα 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. TNFα release negatively correlated [−0.60] with adiponectin release by adipose tissue. The release of TNFα 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. TNFα release over 4 h was enhanced by lipopolysaccharide and inhibited by a cyclooxygenase-2 inhibitor.CONCLUSION: The release of TNFα by adipose tissue of obese humans is primarily due to the nonfat cells present in adipose tissue. TNFα is a short-lived adipokine whose release by human adipose tissue in primary culture correlates with the BMI of the fat donors.

Identification of omentin mRNA in human epicardial adipose tissue: comparison to omentin in subcutaneous, internal mammary artery periadventitial and visceral abdominal depots.

Objective:The purpose of this study was to determine the relative distribution of omentin and visfatin mRNA in human epicardial, peri-internal mammary, upper thoracic, upper abdominal and leg vein subcutaneous adipose tissue as well as the distribution of omentin in the nonfat cells and adipocytes of human omental adipose tissue.Background:Omentin is found in human omentum but not subcutaneous fat. Omentin and visfatin are considered markers of visceral abdominal fat.Research design and methods:The mRNA content of omentin and visfatin was measured by qRT-PCR analysis of fat samples removed from humans undergoing cardiac or bariatric surgery.Results:Omentin mRNA in internal mammary fat was 3.5%, that in the upper thoracic subcutaneous fat was 4.7% while that in the other subcutaneous fat depots was less than 1% of omentin in epicardial fat. The distribution of visfatin mRNA did not vary between the five depots. Omentin mRNA was preferentially expressed in the nonfat cells of omental adipose tissue since the omentin mRNA content of isolated adipocytes was 9% of that in nonfat cells, and similar results were seen for visfatin. The amount of omentin mRNA in differentiated adipocytes was 0.3% and that of visfatin 4% of that in nonfat cells. The amount of omentin mRNA in preadipocytes was virtually undetectable while that of visfatin was 3% of that in freshly isolated nonfat cells from omental adipose tissue.Conclusion:Omentin mRNA is predominantly found in epicardial and omental human fat whereas visfatin mRNA is found to the same extent in epicardial, subcutaneous and omental fat.

Assembly of an SAP97-AKAP79-cAMP-dependent protein kinase scaffold at the type 1 PSD-95/DLG/ZO1 motif of the human β1-adrenergic receptor generates a receptosome involved in receptor recycling and networking

Appropriate trafficking of the β1-adrenergic receptor (β1-AR) after agonist-promoted internalization is crucial for the resensitization of its signaling pathway. Efficient recycling of the β1-AR required the binding of the protein kinase A anchoring protein-79 (AKAP79) to the carboxyl terminus of the β1-AR (Gardner, L. A., Tavalin, S. A., Goehring, A., Scott, J. D., and Bahouth, S. W. (2006) J. Biol. Chem. 281, 33537-33553). In this study we show that AKAP79 forms a complex with the type 1 PDZ-binding sequence (ESKV) at the extreme carboxyl terminus of the β1-AR, which is mediated by the membrane-associated guanylate kinase (MAGUK) protein SAP97. Thus, the PDZ and its associated SAP97-AKAP79 complex are involved in targeting the cyclic AMP-dependent protein kinase (PKA) to the β1-AR. The PDZ and its scaffold were required for efficient recycling of the β1-AR and for PKA-mediated phosphorylation of the β1-AR at Ser312. Overexpression of the catalytic subunit of PKA or mutagenesis of Ser312 to the phosphoserine mimic aspartic acid both rescued the recycling of the trafficking-defective β1-ARΔ PDZ mutant. Thus, trafficking signals transmitted from the PDZ-associated scaffold in the carboxyl terminus of the β1-AR to Ser312 in the 3rd intracellular loop (3rd IC) were paramount in setting the trafficking itinerary of the β1-AR. The data presented here show that a novel β1-adrenergic receptosome is organized at the β1-AR PDZ to generate a scaffold essential for trafficking and networking of the β1-AR.

Adult Epicardial Fat Exhibits Beige Features

CONTEXT Human epicardial fat has been designated previously as brown-like fat. The supraclavicular fat depot in man has been defined as beige coexistent with classical brown based on its gene expression profile. OBJECTIVE The aim of the study was to establish the gene expression profile and morphology of human epicardial and visceral paracardial fat compared with sc fat. SETTING The study was conducted at a tertiary care hospital cardiac center. PATIENTS Epicardial, visceral paracardial, and sc fat samples had been taken from middle-aged patients with severe coronary atherosclerosis or valvular heart disease. INTERVENTIONS Gene expression was determined by reverse transcription-quantitative PCR and relative abundance of the mitochondrial uncoupling protein-1 (UCP-1) by Western blotting. Epicardial tissue sections from patients were examined by light microscopy, UCP-1 immunohistochemistry, and cell morphometry. MAIN OUTCOME MEASURES We hypothesized that epicardial fat has a mixed phenotype with a gene expression profile similar to that described for beige cell lineage. RESULTS Immunoreactive UCP-1 was clearly measurable in each epicardial sample analyzed but was undetectable in each of the 4 other visceral and sc depots. Epicardial fat exhibited high expression of genes for UCP-1, PRDM16, PGC-1α, PPARγ, and the beige adipocyte-specific marker CD137, which were also expressed in visceral paracardial fat but only weakly in sternal, upper abdominal, and lower extremity sc fat. Histology of epicardial fat showed small unilocular adipocytes without UCP-1 immunostaining. CONCLUSION UCP-1 is relatively abundant in epicardial fat, and this depot possesses molecular features characteristic of those found in vitro in beige lineage adipocytes.

Inflammatory Genes in Epicardial Fat Contiguous With Coronary Atherosclerosis in the Metabolic Syndrome and Type 2 Diabetes Changes associated with pioglitazone

OBJECTIVE To determine changes in gene expression in epicardial adipose tissue (EAT) associated with coronary atherosclerosis (CAD) and effects of pioglitazone therapy. RESEARCH DESIGN AND METHODS Genes were quantified by RT-PCR in EAT and thoracic subcutaneous adipose tissue (SAT) obtained during surgery in CAD patients with metabolic syndrome (MS) or type 2 diabetes and control subjects with minimal or no CAD and no MS or type 2 diabetes. RESULTS Increased expression of interleukin-1 receptor antagonist (IL-1Ra) and IL-10, a trend for higher IL-1β, and no change in peroxisome proliferator–activated receptor-γ (PPARγ) was found in EAT from MS or type 2 diabetes. Only PPARγ mRNA was reduced in SAT. Pioglitazone therapy in type 2 diabetes was associated with decreased expression of IL-1β, IL-1Ra, and IL-10 in EAT; decreased IL-10 in SAT; and increased PPARγ in SAT. CONCLUSIONS In MS and type 2 diabetes with CAD, proinflammatory and anti-inflammatory genes were differentially increased in EAT and selectively reduced in association with pioglitazone treatment.

Characterization of the Residues in Helix 8 of the Human β1-Adrenergic Receptor That Are Involved in Coupling the Receptor to G Proteins

Several key amino acids within amphipathic helix 8 of the human β1-adrenergic receptor (β1-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly389 or Arg389 allelic variants of the wild type β1-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type β1-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp382 and Arg384 within the hydrophilic interface might serve as a tethering site for the G protein.

AKAP79-mediated Targeting of the Cyclic AMP-dependent Protein Kinase to the β1-Adrenergic Receptor Promotes Recycling and Functional Resensitization of the Receptor

Resensitization of G protein-coupled receptors (GPCR) following prolonged agonist exposure is critical for restoring the responsiveness of the receptor to subsequent challenges by agonist. The 3′-5′ cyclic AMP-dependent protein kinase (PKA) and serine 312 in the third intracellular loop of the human β1-adrenergic receptor (β1-AR) were both necessary for efficient recycling and resensitization of the agonist-internalized β1-AR (Gardner, L. A., Delos Santos, N. M., Matta, S. G., Whitt, M. A., and Bahouth, S. W. (2004) J. Biol. Chem. 279, 21135-21143). Because PKA is compartmentalized near target substrates by interacting with protein kinase A anchoring proteins (AKAPs), the present study was undertaken to identify the AKAP involved in PKA-mediated phosphorylation of the β1-AR and in its recycling and resensitization. Here, we report that Ht-31 peptide-mediated disruption of PKA/AKAP interactions prevented the recycling and functional resensitization of heterologously expressed β1-AR in HEK-293 cells and endogenously expressed β1-AR in SK-N-MC cells and neonatal rat cortical neurons. Whereas several endogenous AKAPs were identified in HEK-293 cells, small interfering RNA-mediated down-regulation of AKAP79 prevented the recycling of the β1-AR in this cell line. Co-immunoprecipitations and fluorescence resonance energy transfer (FRET) microscopy experiments in HEK-293 cells revealed that the β1-AR, AKAP79, and PKA form a ternary complex at the carboxyl terminus of the β1-AR. This complex was involved in PKA-mediated phosphorylation of the third intracellular loop of the β1-AR because disruption of PKA/AKAP interactions or small interfering RNA-mediated down-regulation of AKAP79 both inhibited this response. Thus, AKAP79 provides PKA to phosphorylate the β1-AR and thereby dictate the recycling and resensitization itineraries of the β1-AR.

Comparison of messenger RNA distribution for 60 proteins in fat cells vs the nonfat cells of human omental adipose tissue

The messenger RNA (mRNA) distribution of 60 proteins was examined in the 3 fractions obtained by collagenase digestion (fat cells and the nonfat cells comprising the tissue remaining after collagenase digestion [matrix] and the stromovascular cells) of omental adipose tissue obtained from morbidly obese women undergoing bariatric surgery. Fat cells were enriched by at least 3-fold as compared with nonfat cells in the mRNAs for retinol binding protein 4, angiotensinogen, adipsin, glutathione peroxidase 3, uncoupling protein 2, peroxisome proliferator-activated receptor gamma, cell death-inducing DFFA-like effector A, fat-specific protein 27, 11beta-hydroxysteroid dehydrogenase 1, glycerol channel aquaporin 7, NADPH:quinone oxidoreductase 1, cyclic adenosine monophosphate phosphodiesterase 3B, glyceraldehyde-3-phosphate dehydrogenase, insulin receptor, and amyloid A1. Fat cells were also enriched by at least 26-fold in the mRNAs for proteins involved in lipolysis such as hormone-sensitive lipase, lipoprotein lipase, adipose tissue triglyceride lipase, and FAT/CD36. The relative distribution of mRNAs in cultured preadipocytes was also compared with that of in vitro differentiated adipocytes derived from human omental adipose tissue. Cultured preadipocytes had far lower levels of the mRNAs for inflammatory proteins than the nonfat cells of omental adipose tissue. The nonfat cells were enriched by at least 5-fold in the mRNAs for proteins involved in the inflammatory response such as tumor necrosis factor alpha, interleukin lbeta, cyclooxygenase 2, interleukin 24, interleukin 6, and monocyte chemoattractant protein 1 plus the mRNAs for osteopontin, vaspin, endothelin, angiotensin II receptor 1, butyrylcholinesterase, lipocalin 2, and plasminogen activator inhibitor 1. The cells in the adipose tissue matrix were enriched at least 3-fold as compared with the isolated stromovascular cells in the mRNAs for proteins related to the inflammatory response, as well as osteopontin and endothelial nitric oxide synthase. We conclude that the mRNAs for inflammatory proteins are primarily present in the nonfat cells of human omental adipose tissue.

Compartmentalized Cyclic Adenosine 3,5-Monophosphate at the Plasma Membrane Clusters PDE3A and Cystic Fibrosis Transmembrane Conductance Regulator into Microdomains

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.