Adipose tissue plays a pivotal role in metabolic, physiologic, and endocrine homeostasis [68,69]. This highly specialized tissue of mesenchymal origin is comprised of multiple cell types held loosely together in a collagen matrix. The predominant cell type is the terminally differentiated adipocyte, a very large cell whose size can be dramatically altered under various nutritional states. The stroma, also called the stromal vascular fraction (SVF), contains pluripotent stem cells, preadipocytes, endothelial cells, pericytes, mast cells, fibroblasts, and hematopoietic cells, mainly macrophages.
Two types of adipose tissue are recognized: white adipose tissue (WAT), which stores energy in the forms of lipids, and brown adipose tissue (BAT), which generates heat by uncoupling the mitochondrial aerobic respiration chain that normally results in ATP generation . Human WAT is found in specific anatomical depots which differ in morphology and functions and are grossly classified as abdominal visceral (vis) and subcutaneous (sc) fat. The relative distribution of these depots determines the gender-dependent body shape and affects the development of metabolic syndrome (discussed later in this chapter). Adipose tissue also functions as an important endocrine organ that produces a multitude of adipokines that target many organs and modulate a wide range of metabolic pathways.
Non-shivering thermogenesis in brown adipocytes is activated by beta-AR . DA also affects thermogenesis in brown adipocytes, except that both stimulatory and inhibitory actions have been reported. An early study  found that similar to NE, DA increased oxygen consumption in rat BAT explants. Another group, using isolated rat brown adipocytes, reported that DA antagonized the beta-AR stimulation of glycerol and nonesterified fatty acid release . Both studies, however, used very high micromolar concentrations of DA. This issue was rectified in a later study employing immortalized murine brown adipocytes . Within 24 h of treatment with low nanomolar doses of DA or a DIR agonist, there were increases in oxygen consumption, mitochondrial membrane potential, and uncoupling protein 1 (UCP1) levels, implicating the activation of heat production by the cells.
An early study reported that DA stimulated glucose uptake in rodent adipocytes , but attributed this action to the activation of beta-AR by DA. A later study compared the lipolytic action of adrenergic agonists and antagonists as well as that of DA in visceral adipocytes harvested from betal/ beta2/beta3-AR triple-knockout (beta-less) mice . The authors found residual lipotytic activity of most of these agents, which they attributed to an unknown G protein-coupled receptor (GPCR) with low affinity for catecholamines. Because expression of DAR in adipocytes was not known to these authors at that time and all agents were tested at 10-100 pM, activation of lipolysis in the beta-less adipocytes was probably mediated by DAR.
In murine 3T3-L1 adipocytes, the D2R agonist bromocriptine decreased expression of both adipogenic activators and lipogenic target genes . Bromocriptine also reduced intracellular nitric oxide formation and expression of pro-inflammatory genes, reflecting attenuated pro-inflammatory responses. Treatment of D2R-deficient 3T3-L1 with bromocriptine significantly decreased lipogenic activity, whereas treatment with yohimbine, an cx2-AR inhibitor, showed no reduction in lipogenic activity. The authors concluded that the bromocriptine-induced attenuation of adipogenesis and lipogenesis in 3T3-L1 cells was mediated by cx2-AR rather than by D2R. Yet, bromocriptine was used at 10-50 pM doses, raising the possibility of cross reactivity with ARs.
The discovery that human adipocytes express functional DAR was the consequence of our pursuit of the regulation of extrapituitary PRL . Unique to humans, PRL is expressed in many extrapituitary sites, including the endometrium, myometrium, decidua, immune cells, brain, breast, prostate, skin and adipose tissue. Upon monitoring PRL release from sc and vis adipose explants from obese and lean patients, we unexpectedly noticed a gradual, time-related increase in PRL release during incubation, suggesting a removal from tonic inhibition . Because a similar rise in PRL release occurs when pituitary lactotrophs are removed from hypothalamic DA inhibition, we wondered if DA also serves as an inhibitor of adipocyte PRL.
Subsequent!)', we used real-time polymerase chain reaction (RT-PCR) and Western blotting and detected expression of several types of DAR in human adipocytes . In addition, we found that both DA and DA-S, the major circulating form of DA in humans, inhibited adipocyte PRL release (see Figure 6.4 in Chapter 6). Although synthesis of DA by human adipocytes has not been determined in this study, others reported that isolated rat mesenteric adipocytes, as well SVF, expressed TH, DBH, and PNMT and were fully capable of de novo synthesis of catecholamines, including small amounts of DA . In addition to the local production, DA can be made available to adipocytes by uptake from extracellular depots secreted by neighboring resident immune cells.
As determined by RT-PCR and Western blotting, DIR, D2R, and D4R, and to a lesser extent D5R, were expressed in human adipose tissue explants, primary adipocytes, and two adipocyte cell lines, SW872 and LSI4 . SW872 is a liposarcoma-derived cell line from the ATCC. LSI4 is a spontaneously immortalized adipocyte cell line that we cloned from a metastatic liposar- coma . The functionality of adipocyte D2R was confirmed by the inhibition of PRL release by both DA and bromocriptine, a D2R agonist, and its blockade by raclopride, a D2R antagonist . The functionality of D1R was confirmed by the induction of ERK1 /2 phosphotylation by DA, with a delayed effect on Akt.
Notably, as illustrated in Figure 8.8, Panels A and B, expression of DIR and D2R was differentially altered during early adipogenesis, with DIR increasing while D2R decreasing on day 3 of adipogenesis, before the appearance of differentiated adipocytes. This suggested a potential regulation of DAR by transcription factors such as peroxisome proliferator-activated receptor gamma (PPARy) and CCAAT-enhancer-binding protein (cEBPa), which are critical for preadipocyte differentiation and whose expression increases during the first few days of adipogenesis . Indeed, we have identified a PPARy binding site in the promoters of both DIR and D2R and a C/EBP binding site in DIR (Figure 8.8C).
Expression of serotonin receptors 5-HTlaR, 5-HT2aR, and 5-HT2cR, considered the prime targets of atypical antipsychotics (AAPs) in the brain, was also examined in this study. The 5-HT1 aR was low to undetectable in adipose tissue and preadipocytes but showed a strong signal in differentiated adipocytes. The 5-HT2aR was detected in adipose tissue and was lower in differentiated than in proliferating adipocytes. The 5-HT2cR was undetectable in either adipose tissue or adipocytes.
In addition to inhibiting PRL release, DA altered the release of leptin, adiponectin, and interleukin-6 (IL-6) from human adipocytes . Leptin, whose production is proportional to adipose tissue mass, plays an important role in the regulation of energy homeostasis, neuroendocrine and immune functions and in glucose, lipid and bone metabolism. Leptin administration to humans reduces appetite, while chronic leptin deficiency causes extreme obesity. Adiponectin protects against metabolic syndrome by virtue of its insulin-sensitizing, anti-inflammatory, and anti-atherogenic activities. IL-6 is a pro-inflammatory cytokine associated with the low level of inflammation that accompanies obesity. High serum IL-6 levels increase the production of C-reactive protein and can lead to coronary heart disease and atherosclerosis.
The effects of low (1 nM) and high (100 nM) doses of DA on leptin release were examined using human sc adipose explants, isolated mature adipocytes, and differentiated primary adipocytes . Leptin release was inhibited 40%-80% by either dose of DA and also by the D1R/D5R agonist, SKF38393 (SKF), suggesting an action via DIR-like. Both DA and SKF caused a 60%-80% increase in adiponectin release from differentiated primary adipocytes and an increase in IL-6 release from proliferating primary preadipocytes. Others have reported that DA exerted dose-related reduction of leptin gene expression and release from adipocytes harvested from obese hypertensive patients . The authors proposed that DA deficiency contributes to metabolic disorders linked to the hyperleptinemia in these patients.
In a recent study using 3T3-L1 adipocytes, quinpirole, a D2R agonist, increased both protein and mRNA expression of leptin and IL-6, but not adiponectin and visfatin, and also increased the mRNA expression of tumor necrosis factor alpha (TNF-a), monocyte chemoattractant protein-1 (MCP1), and nuclear factor kappa В p50 pathway (NF-кВ-рбО) . Acute quinpirole treatment in C57BL/6J mice increased serum leptin concentration and leptin mRNA in visceral adipose tissue but not in subcutaneous adipose, confirming the stimulatory effect of D2R on leptin in vivo. The authors concluded that the stimulation of D2R increases leptin production and may have a tissue-specific pro-inflammatory effect in adipocytes.
Figure 8.8 Panel A: Changes in D1R and D2R expression during differentiation of human preadipocytes. Panel B: Changes in the structure of the adipocytes in selected days of differentiation. Panel C: Identification of putative binding sites for various transcription factors in the promoters of D1R and D2R. (Redrawn and modified from Borcherding, D.C. et al. PLoS One, 6, e25537,2011.)
Figure 8.9 Panel A shows inhibition of leptin release by antipsychotics. Con: control; Olan: olanzapine; Zipr: ziprasidone; Halo: haloperidol. Panel В shows inhibition of leptin release by the serotonin ligands 5HTP and 5HT. Each value is a mean +/-SEM of 5 determination. * p<0.05. (Unpublished data from the author's laboratory.)
The effects of selected AAPs on leptin release from isolated primaiy human adipocytes were also determined in our laboratory (Figure 8.9A). Olanzapine, ziprasidone and haloperidol suppressed leptin release in a nonmonotonic fashion. The lack of linearity and the variable responses were likely due to concomitant activation or inhibition of DAR and/or serotonergic receptor subtypes by the increasing concentrations of the AAP. Figure 8.9B also shows that both 5-HT and its precursor 5-HTP inhibited leptin release from sc explants, confirming not only the functionality of serotonergic receptors in human adipocytes but also demonstrating the capacity of human adipose tissue for de novo synthesis of serotonin from its precursor 5HTP. In addition to the regulation of PRL release from the adipocytes, our data showed that AAPs differentially affect lipolysis, lipogenesis and preadipocyte differentiation. This likely occurs by differentially activating various DAR and serotonergic receptors.
A conceptual model of the potential interactions between DIR, D2R and 5HTR in the regulation of the adipocytes is presented in Figure 8.10. The various receptor types may work in a cooperative or in an antagonistic manner to regulate lipogenesis, lipolysis, adipogenesis as well as adipokine release, depending on the relative expression of each receptor and availability of endogenous ligands and administered drugs.