John N. Fain

Release of interleukins and other inflammatory cytokines by human adipose tissue is enhanced in obesity and primarily due to the nonfat cells.

The white adipose tissue, especially of humans, is now recognized as the central player in the mild inflammatory state that is characteristic of obesity. The question is how the increased accumulation of lipid seen in obesity causes an inflammatory state and how this is linked to the hypertension and type 2 diabetes that accompanies obesity. Once it was thought that adipose tissue was primarily a reservoir for excess calories that were stored in the adipocytes as triacylglycerols. In times of caloric deprivation these stored lipids were mobilized as free fatty acids and the insulin resistance of obesity was attributed to free fatty acids. It is now clear that in humans the expansion of adipose tissue seen in obesity results in more blood vessels, more connective tissue fibroblasts, and especially more macrophages. There is an enhanced secretion of some interleukins and inflammatory cytokines in adipose tissue of the obese as well as increased circulating levels of many cytokines. The central theme of this chapter is that human adipose tissue is a potent source of inflammatory interleukins plus other cytokines and that the majority of this release is due to the nonfat cells in the adipose tissue except for leptin and adiponectin that are primarily secreted by adipocytes. Human adipocytes secrete at least as much plasminogen activator inhibitor-1 (PAI-1), MCP-1, interleukin-8 (IL-8), and IL-6 in vitro as they do leptin but the nonfat cells of adipose tissue secrete even more of these proteins. The secretion of leptin, on the other hand, by the nonfat cells is negligible. The amount of serum amyloid A proteins 1 & 2 (SAA 1 & 2), haptoglobin, nerve growth factor (NGF), macrophage migration inhibitory factor (MIF), and PAI-1 secreted by the adipocytes derived from a gram of adipose tissue is 144%, 75%, 72%, 37%, and 23%, respectively, of that by the nonfat cells derived from the same amount of human adipose tissue. However, the release of IL-8, MCP-1, vascular endothelial growth factor (VEGF), TGF-beta1, IL-6, PGE(2), TNF-alpha, cathepsin S, hepatocyte growth factor (HGF), IL-1beta, IL-10, resistin, C-reactive protein (CRP), and interleukin-1 receptor antagonist (IL-1Ra) by adipocytes is less than 12% of that by the nonfat cells present in human adipose tissue. Obesity markedly elevates the total release of TNF-alpha, IL-6, and IL-8 by adipose tissue but only that of TNF-alpha is enhanced in adipocytes. However, on a quantitative basis the vast majority of the TNF-alpha comes from the nonfat cells. Visceral adipose tissue also releases more VEGF, resistin, IL-6, PAI-1, TGF-beta1, IL-8, and IL-10 per gram of tissue than does abdominal subcutaneous adipose tissue. In conclusion, there is an increasing recognition that adipose tissue is an endocrine organ that secretes leptin and adiponectin along with a host of other paracrine and endocrine factors in addition to free fatty acids.

Role of phosphatidylinositol turnover in alpha1 and of adenylate cyclase inhibition in alpha2 effects of catecholamines

Abstract Ligand binding and pharmacological studies have indicated that alpha-adrenergic receptors can be divided into alpha1 and alpha2. We suggest that alpha1 receptors mediate those metabolic effects of alpha catecholamines which involve phosphatidylinositol turnover and the release of bound intracellular Ca2+ as well as the entry of extracellular Ca2+. In contrast, alpha effects of catecholamines are due to non-specific inhibition of adenylate cyclase through a mechanism independent of Ca2+. A similar classification for the effects of both histamine and serotonin suggests that they have separate type 1 or alpha receptors for Ca2+ dynamics which are different from type 2 or beta receptors which regulate adenylate cyclase. There is a significant correlation between hormone effects on phosphatidylinositol turnover and elevation of intracellular Ca2+. The available data suggest that the turnover of membrane-bound phosphatidylinositol is involved in Ca2+ gating in rat hepatocytes, rat and hamster adipocytes and blowfly salivary glands. In hamster adipocytes adenylate cyclase activity is also inhibited by alpha2 catecholamines through a Ca2+ independent mechanism.

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.

Release of Inflammatory Mediators by Human Adipose Tissue Is Enhanced in Obesity and Primarily by the Nonfat Cells: A Review

This paper considers the role of putative adipokines that might be involved in the enhanced inflammatory response of human adipose tissue seen in obesity. Inflammatory adipokines [IL-6, IL-10, ACE, TGFβ1, TNFα, IL-1β, PAI-1, and IL-8] plus one anti-inflammatory [IL-10] adipokine were identified whose circulating levels as well as in vitro release by fat are enhanced in obesity and are primarily released by the nonfat cells of human adipose tissue. In contrast, the circulating levels of leptin and FABP-4 are also enhanced in obesity and they are primarily released by fat cells of human adipose tissue. The relative expression of adipokines and other proteins in human omental as compared to subcutaneous adipose tissue as well as their expression in the nonfat as compared to the fat cells of human omental adipose tissue is also reviewed. The conclusion is that the release of many inflammatory adipokines by adipose tissue is enhanced in obese humans.

Regulation of adenylate cyclase by adenosine.

SummaryAdenosine may well be as important in the regulation of adenylate cyclase as hormones. Sattin and Rall first demonstrated in 1970 that adenosine was a potent stimulator of adenylate cyclase in the brain. However, adenosine is an equally potent inhibitor of adenylate cyclase in other cells such as adipocytes. The concentration of adenosine required for this regulation of adenylate cyclase is in the nanomolar range (10 to 100 nm). Both the inhibitory and stimulatory effects of low concentrations of adenosine on adenylate cyclase are antagonized by methylxanthines. This antagonism of adenosine action may account for all or part of the effects of methyl xanthines on cyclic AMP levels in many tissues. Adenosine appears to be a particularly important endogenous regulator of adenylate cyclase in brain, smooth muscle and fat cells. Under conditions in which intracellular AMP rises, adenosine formation and release is accelerated. In addition to its direct effects on adenylate cyclase, adenosine (at higher concentrations approaching millimolar) exerts multiple effects on cellular metabolism as a result of its intracellular metabolism and especially conversion to nucleotides.The effects of nanomolar concentrations of adenosine on adenylate cyclase are mediated through an adenosine site possessing strict structural specificity for the ribose moiety of the molecule (the “R” adenosine site) which is presumably located on the external surface of the plasma membrane. In brain, lung, platelets, bone, lymphocytes, skin, adrenals, Leydig tumors, and coronary arteries adenosine stimulates adenylate cyclase via this site. However, in rat adipocytes, brain astroblasts and ventricular myocardium adenosine inhibits adenylate cyclase through the “R” or adenosine site. Although the “R” site requires an intact ribose moiety, adenosine analogs modified in the purine ring such as N6-phenylisopropyladenosine appear to be potent agonists for this site. All effects of adenosine mediated via the “R” site are competitively antagonized by methyl xanthines.The effects of micromolar concentrations of adenosine appear to be mediated via a site with strict structural specificity with respect to the purine moiety of the molecule (the “P” or adenine adenosine site). This “P” site is postulated to be located on the intracellular face of the plasma membrane and mediates the effects of adenosine due to conversion of adenosine to 5′-AMP or perhaps other nucleotides. The effects of high concentrations of adenosine are always inhibitory to adenylate cyclase activity, are readily demonstrated in broken cell preparations, and are unaffected by methylxanthines. An intact purine ring is required for these adenosine effects but modifications of the ribose moiety of the molecule generally increases the potency of the analog. A prime example is 2′,5′-dideoxyadenosine, which is the most potent known “R”-site specific adenosine analog.We propose a unitary model which explains both the stimulatory and inhibitory effects of low concentrations of adenosine on adenylate cyclase. In brief, adenylate cyclase is postulated to exist in three interconvertible activity states: (i) an inactive state (E0); (ii) a GTP-liganded state with high activity (EGTP); and (iii) a GDP-liganded state (EGDP) which is inactive in cells where adenosine stimulates adenylate cyclase, but active in cells where adenosine inhibits adenylate cyclase. We postulate that the enzyme cycles through these states in the following manner: the E0 state binds GTP and forms the EGTP state; hydrolysis of bound GTP converts the EGTP to the EGDP state; and release of bound GDP converts EGDP to the E0 state. The E0 state is the only form of the enzyme which can be stimulated by either hormones or GTP and its formation from the EGDP state is rate-limiting in this cycle. The conversion of EGDP to E0 regulates the ability of hormones and GTP to activate adenylate cyclase and is postulated to be adenosine sensitive.In cells where both EGDP and E0 states are inactive, adenosine stimulates adenylate cyclase activity. In cells where E0 is inactive, but EGDP is active, adenosine inhibits adenylate cyclase activity. In addition we suggest that in cells where adenosine inhibits adenylate cyclase activity (cells postulated to have an EGDP state which is active) high concentrations of GTP favor accumulation of the enzyme in EGDP and thus are inhibitory to activity. Prostaglandins may also regulate adenylate cyclase in a manner similar to that described above for adenosine.We conclude that adenosine is an important regulator of adenylate cyclase whose role has only been appreciated recently. Further studies are warranted on both its binding to cells and mechanisms by which it regulates adenylate cyclase.

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.

Regulation of phosphoinositide breakdown by guanine nucleotides

Phosphoinositide hydrolysis is coupled to receptor systems involved in the elevation of cytosolic Ca2+ and activation of protein kinase C. In cell-free systems, guanine nucleotides are required to transduce the effects of receptor activation to phosphoinositide breakdown. Non-hydrolyzable guanine nucleotides stimulate phosphoinositide breakdown in permeabilized cells as well as membranes prepared from salivary glands, GH3 cells, neutrophils, hepatocytes and cerebral cortical tissue. In blowfly salivary gland membranes, 5-hydroxytryptamine stimulates a guanine-nucleotide dependent breakdown of both endogenous and exogenous phosphoinositide substrate through activation of phospholipase C. These data suggest that a GTP-binding protein modulates phospholipase C activity. The identity of this GTP-binding protein has not been established but may resemble other regulatory GTP-binding proteins which have been identified as transducing proteins in a variety of receptor systems.

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.

Pharmacological Characterizations of Adrenergic Receptors in Human Adipocytes

Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [(3)H]dihydroalprenolol binding (beta adrenergic) B(max) 280 fmol/mg protein, K(D) 0.38 nM; [(3)H]para-aminoclonidine binding (alpha-2 adrenergic) B(max) 166 fmol/mg protein, K(D) 0.49 nM; [(3)H]WB 4101 binding (alpha-1 adrenergic) B(max) 303 fmol/mg protein, K(D) 0.86 nM. In adipocytes from subcutaneous adipose tissue, [(3)H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors. Beta and alpha-2 adrenergic receptors appeared to be positively and negatively coupled to adenylate cyclase, respectively. Cells or cell membranes were incubated with epinephrine (10 muM) alone and in combination with the antagonists yohimbine (alpha-2) and prazosin (alpha-1). Epinephrine alone prompted a modest increase in adenylate cyclase activity, cyclic AMP, and glycerol release, an index of lipolysis. Yohimbine (0.1 muM) greatly enhanced these actions whereas prazosin was without effect. The beta agonist, isoproterenol, stimulated glycerol release, whereas the alpha-2 agonist, clonidine, inhibited lipolysis and cyclic AMP accumulation. To assess further alpha-1 receptors, cells were incubated with [(32)P]phosphate and epinephrine (10 muM) alone and in combination with prazosin and yohimbine. Epinephrine alone caused a three- to fourfold increase in (32)P incorporation into phosphatidylinositol. Prazosin (0.1 muM) blocked this action whereas yohimbine (0.1 muM) was without effect. Thus, in a homogeneous cell preparation, the human adipocyte appears to have three different adrenergic receptors, each of which is coupled to a distinct biochemical response.

Role of alpha1 adrenoceptors in the turnover of phosphatidylinositol and of alpha2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

Abstract The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin⪢phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha1 adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ⪢prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha2 adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha1 and alpha2 adrenoceptors but about 90% of the binding sites were alpha2. These data support the hypothesis that alpha2 effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha1 adrenoceptor activation.