Eric R. Hugo

Bisphenol a at Environmentally Relevant Doses Inhibits Adiponectin Release from Human Adipose Tissue Explants and Adipocytes

Background The incidence of obesity has risen dramatically over the last few decades. This epidemic may be affected by exposure to xenobiotic chemicals. Bisphenol A (BPA), an endocrine disruptor, is detectable at nanomolar levels in human serum worldwide. Adiponectin is an adipocyte-specific hormone that increases insulin sensitivity and reduces tissue inflammation. Thus, any factor that suppresses adiponectin release could lead to insulin resistance and increased susceptibility to obesity-associated diseases. Objectives In this study we aimed to compare a) the effects of low doses of BPA and estradiol (E2) on adiponectin secretion from human breast, subcutaneous, and visceral adipose explants and mature adipocytes, and b) expression of putative estrogen and estrogen-related receptors (ERRs) in these tissues. Methods We determined adiponectin levels in conditioned media from adipose explants or adipocytes by enzyme-linked immunosorbant assay. We determined expression of estrogen receptors (ERs) α and β, G-protein–coupled receptor 30 (GPR30), and ERRs α, β, and γ by quantitative real-time polymerase chain reaction. Results BPA at 0.1 and 1 nM doses suppressed adiponectin release from all adipose depots examined. Despite substantial variability among patients, BPA was as effective, and often more effective, than equimolar concentrations of E2. Adipose tissue expresses similar mRNA levels of ERα, ERβ, and ERRγ, and 20- to 30-fold lower levels of GPR30, ERRα, and ERRβ. Conclusions BPA at environmentally relevant doses inhibits the release of a key adipokine that protects humans from metabolic syndrome. The mechanism by which BPA suppresses adiponectin and the receptors involved remains to be determined.

Effects of bisphenol A on adipokine release from human adipose tissue: Implications for the metabolic syndrome.

Bisphenol A (BPA) is one of the most prevalent and best studied endocrine disruptors. After years of exposure to consumer products containing BPA, most individuals tested have circulating BPA at the low nanomolar levels. In addition to its well documented actions on the reproductive system, BPA exerts a wide variety of metabolic effects. This review summarizes recent findings on the ability of BPA, at environmentally relevant doses, to inhibit adiponectin and stimulate the release of inflammatory adipokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) from human adipose tissue. Expression of several classical and non-classical estrogen receptors in human adipose tissue raises the possibility of their involvement as mediators of BPA actions. The implications of these observations to the obesity-related metabolic syndrome and its sequelae are discussed.

Focus on prolactin as a metabolic hormone

New information about the effects of prolactin (PRL) on metabolic processes warrants re-evaluation of the overall metabolic actions of PRL. PRL affects metabolic homeostasis by regulating key enzymes and transporters that are associated with glucose and lipid metabolism in several target organs. In the lactating mammary gland, PRL increases the production of milk proteins, lactose and lipids. In adipose tissue, PRL generally suppresses lipid storage and adipokine release. PRL supports the growth of pancreatic islets, stimulates insulin secretion and increases citrate production in the prostate. A specific case is made for PRL in the human breast and adipose tissue, where it acts as a circulating hormone and an autocrine or paracrine factor. Although the overall effects of PRL on body composition are modest and species specific, PRL might be involved in the manifestation of insulin resistance.

Low dose effects of bisphenol A

In 2007, a group of experts critically analyzed hundreds of publications on bisphenol A (BPA), including the evidence for low dose effects. Here, we have updated these evaluations to determine the strength of the evidence for low dose effects of BPA. Based on the cut-offs for “low doses” established previously (i.e., the lowest observed adverse effect level [LOAEL], or 50 mg/kg/day for mammalian studies), we identified more than 450 low dose studies. Using an integrative approach, we examined five endpoints in depth that had evidence from two or more study types (in vitro, in vivo laboratory animal, and human). Based on all available studies, we are confident that consistent, reproducible, low dose effects have been demonstrated for BPA. We conclude that the doses that reliably produce effects in animals are 1–4 magnitudes of order lower than the current LOAEL of 50 mg/kg/day and many should be considered adverse.

Adipocyte prolactin: regulation of release and putative functions.

Pituitary‐derived prolactin (PRL) is a well‐known regulator of the lactating mammary gland. However, the recent discovery that human adipose tissue produces PRL as well as expresses the PRL receptor (PRLR) highlights a previously unappreciated action of PRL as a cytokine involved in adipose tissue function. Biologically active PRL is secreted by all adipose tissue depots examined: breast, visceral and subcutaneous. The expression of adipose PRL is regulated by a non‐pituitary, alternative superdistal promoter. PRL expression and release increases during early pre‐adipocyte differentiation and is stimulated by cyclic AMP activators, including β adrenergic receptor agonists. PRL release from subcutaneous adipose explants is attenuated during obesity, suggesting that adipose PRL production is altered by the metabolic state. Several lines of evidence indicate that PRL suppresses lipid storage as well as the release of adipokines such as adiponectin, interleukin‐6 and possibly leptin. PRL has also been implicated in the regulation of adipogenesis. A newly developed PRL‐secreting human adipocyte cell line, LS14, should allow comprehensive examination of the regulation and function of adipocyte‐derived PRL. Collectively, these studies raise the prospect that PRL affects energy homeostasis through its action as an adipokine and is involved in the manifestation of insulin resistance.

Dopamine Receptors in Human Adipocytes: Expression and Functions

Introduction Dopamine (DA) binds to five receptors (DAR), classified by their ability to increase (D1R-like) or decrease (D2R-like) cAMP. In humans, most DA circulates as dopamine sulfate (DA-S), which can be de-conjugated to bioactive DA by arylsulfatase A (ARSA). The objective was to examine expression of DAR and ARSA in human adipose tissue and determine whether DA regulates prolactin (PRL) and adipokine expression and release. Methods DAR were analyzed by RT-PCR and Western blotting in explants, primary adipocytes and two human adipocyte cell lines, LS14 and SW872. ARSA expression and activity were determined by qPCR and enzymatic assay. PRL expression and release were determined by luciferase reporter and Nb2 bioassay. Analysis of cAMP, cGMP, leptin, adiponectin and interleukin 6 (IL-6) was done by ELISA. Activation of MAPK and PI3 kinase/Akt was determined by Western blotting. Results DAR are variably expressed at the mRNA and protein levels in adipose tissue and adipocytes during adipogenesis. ARSA activity in adipocyte increases after differentiation. DA at nM concentrations suppresses cAMP, stimulates cGMP, and activates MAPK in adipocytes. Acting via D2R-like receptors, DA and DA-S inhibit PRL gene expression and release. Acting via D1R/D5R receptors, DA suppresses leptin and stimulates adiponectin and IL-6 release. Conclusions This is the first report that human adipocytes express functional DAR and ARSA, suggesting a regulatory role for peripheral DA in adipose functions. We speculate that the propensity of some DAR-activating antipsychotics to increase weight and alter metabolic homeostasis is due, in part, to their direct action on adipose tissue.

Prolactin Release by Adipose Explants, Primary Adipocytes, and LS14 Adipocytes

BACKGROUND Prolactin (PRL) is a multifunctional hormone produced in humans by both pituitary and extrapituitary sites, including adipose tissue. OBJECTIVES Our objectives were to: 1) compare PRL secretion by sc and visceral adipose explants and mature adipocytes from obese and nonobese patients; and 2) examine the effects of insulin and selected cytokines on PRL gene expression and release from primary adipocytes and LS14 adipocytes. DESIGN AND SUBJECTS Adipose tissue was obtained from morbidly obese [body mass index (BMI) > 40 kg/m(2)] and nonobese (BMI <30 kg/m(2)) patients. Explants and isolated mature adipocytes were incubated for 10 d. Primary adipocytes or LS14 cells were used before or after differentiation and incubated with the test compounds for 24 h. PRL release was analyzed by a bioassay, and PRL expression was determined by real-time PCR. RESULTS PRL release from explants and mature adipocytes increased in a time-dependent manner indicating removal from inhibition. Visceral explants from obese patients showed higher PRL release than that from sc explants; both types of explants from nonobese patients released similar amounts of PRL. Analysis of data from 50 patients revealed an inverse relationship between PRL release from sc depots and BMI. Insulin suppressed PRL expression and release from differentiated adipocytes but moderately stimulated PRL release from nondifferentiated cells. The cAMP elevating compound forskolin increased PRL release in both cell types. CONCLUSIONS PRL should be recognized as an important adipokine whose release is regulated by insulin and is affected by obesity in a depot-specific manner.

Insulin Stimulates Interleukin-6 Expression and Release in LS14 Human Adipocytes through Multiple Signaling Pathways

IL-6 is an important cytokine that regulates both immune and metabolic functions. Within adipose tissue, preadipocytes produce significant amounts of IL-6, but little is known about the factors or mechanisms that regulate IL-6 production in these cells. Using LS14, a newly developed human adipocyte cell line, our objective was to determine the mechanisms by which insulin stimulates IL-6 production and release in preadipocytes. Insulin increased IL-6 gene expression and secretion in a time- and dose-dependent manner. Insulin decreased cyclic AMP (cAMP) but increased cyclic GMP (cGMP) levels, and IL-6 expression/release was stimulated by a cGMP analog. The stimulatory effect of insulin and cGMP was abrogated by a specific inhibitor of protein kinase G (cyclic GMP-dependent protein kinase). Both insulin and cGMP rapidly induced phosphorylation of cAMP response element binding protein. Insulin also activated the MAPK signaling pathway, and its blockade prevented the insulin-stimulated increases in IL-6 cell content and release, but not IL-6 gene expression. Although inhibition of the proteosome increased IL-6 cell content and release, proteosome activity was unaffected by insulin. These data suggest that the stimulatory effects of insulin on IL-6 release involve several interrelated components: transcription, intracellular releasable pool, and secretion, which are differentially regulated and, thus, determine the size of the releasable pool of IL-6. Insulin-induced IL-6 gene expression is mediated by cGMP/cyclic GMP-dependent protein kinase/cAMP response element binding protein, whereas MAPK is involved in the insulin-stimulated IL-6 synthesis/release.

Unexploited therapies in breast and prostate cancer: blockade of the prolactin receptor.

Breast and prostate cancers are hormone-sensitive malignancies that afflict millions of women and men. Although prolactin (PRL) is known as a survival factor that supports tumor growth and confers chemoresistance in both cancers, its precise role in these tumors has not been studied extensively. Growth hormone and placental lactogen also bind PRL receptor (PRLR) and mimic some of the actions of PRL. Blockade of the PRLR represents a novel treatment for patients with advanced breast or prostate cancer with limited therapeutic options. This review discusses different approaches for generating PRLR antagonists. Emphasis is placed on technological advances which enable high-throughput screening for small molecule inhibitors of PRLR signaling that could serve as oral medications.

Calcium sensing receptor activation elevates proinflammatory factor expression in human adipose cells and adipose tissue.

The proinflammatory status of adipose tissue has been linked to the metabolic and cardiovascular consequences of obesity. Human adipose cells express the calcium sensing receptor (CaSR), and its expression is elevated in inflammatory states, such as that associated with obesity. Given the CaSRs association with inflammation in other tissues, we evaluated its role elevating the adipose expression of inflammatory factors. The CaSR activation by the calcimimatic cinacalcet (5μM) in adipose tissue and in vitro cultured LS14 adipose cells elicited an elevation in the expression of the proinflammatory cytokines IL6, IL1β, TNFα, and the chemoattractant CCL2. This was in part reverted by SN50, an inhibitor of the inflammatory mediator nuclear factor kappa B (NFκB). Our observations suggest that CaSR activation elevates cytokine and chemokine production, partially mediated by NFκB. These findings support the relevance of the CaSR in the pathophysiology of obesity-induced adipose tissue dysfunction, with an interesting potential for pharmacological manipulation.