Ana Kilić

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells—so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine–A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Increased cGMP promotes healthy expansion and browning of white adipose tissue

With more than half a billion individuals affected worldwide, obesity has reached pandemic proportions. Development of “brown‐like” or “brite” adipocytes within white adipose tissue (WAT) has potential antiobesity and insulin‐sensitizing effects. We investigated the role of cyclic GMP (cGMP) signaling, focusing on cGMP‐dependent protein kinase I (PKGI) in WAT. PKGI is expressed in murine WAT, primary adipocytes, and 3T3‐L1. Treatment of adipocytes with cGMP resulted in increased adipogenesis, with a 54% increase in expression of peroxisome proliferator‐activated receptor‐γ. Lentiviral overexpression of PKGI further increased adipogenesis, whereas loss of PKGI significantly reduced adipogenic differentiation. In addition to adipogenic effects, PKGI had an antihypertrophic and anti‐inflammatory effect via RhoA phosphorylation and reduction of proinflammatory adipokine expression. Moreover, PKGI induced a 4.3‐fold increase in abundance of UCP‐1 and the development of a brown‐like thermogenic program in primary adipocytes. Notably, treatment of C57BL/6 mice with phosphodiesterase inhibitor sildenafil (12 mg/kg/d) for 7 d caused 4.6‐fold increase in uncoupling protein‐1 expression and promoted establishment of a brown fat cell‐like phenotype (“browning”) of WAT in vivo. Taken together, PKGI is a key regulator of cell size, adipokine secretion and browning of white fat depots and thus could be a valuable target in developing novel treatments for obesity.—Mitschke, M. M., Hoffmann, L. S., Gnad, T., Scholz, D., Kruithoff, K., Mayer, P., Haas, B., Sassmann, A., Alexander Pfeifer, A, Kilić, A. Increased cGMP promotes healthy expansion and browning of white adipose tissue. FASEB J. 27, 1621–1630 (2013). www.fasebj.org

Combined inhibition of PI3Kβ and PI3Kγ reduces fat mass by enhancing α-MSH–dependent sympathetic drive

Blocking the activity of two PI3K isoforms in the brain may be a strategy for treating obesity. Making the Brain Promote Fat Loss Obesity is associated with diabetes and metabolic syndrome and is a growing global problem. Signals from the brain regulate whole body metabolism and can trigger adipose tissue to burn fat. Perino et al. found that mice expressing catalytically inactive forms of two phosphatidylinositol 3-kinases (PI3Kβ and PI3Kγ) were leaner, burned more fat, and expended more energy than their wild-type counterparts. Fat loss also occurred in mice that received inhibitors of PI3Kβ and PI3Kγ delivered specifically into the brain. Thus, drugs that block the activity of both PI3Kβ and PI3Kγ in the brain could be developed as a treatment to fight obesity. Obesity is defined as an abnormal increase in white adipose tissue and has become a major medical burden worldwide. Signals from the brain control not only appetite but also energy expenditure, both of which contribute to body weight. We showed that genetic or pharmacological inhibition of two phosphatidylinositol 3-kinases (PI3Kβ and PI3Kγ) in mice reduced fat mass by promoting increased energy expenditure. This effect was accompanied by stimulation of lipolysis and the acquisition of the energy-burning characteristics of brown adipocytes by white adipocytes, a process referred to as “browning.” The browning of the white adipocytes involved increased norepinephrine release from the sympathetic nervous system. We found that PI3Kβ and PI3Kγ together promoted a negative feedback loop downstream of the melanocortin 4 receptor in the central nervous system, which controls appetite and energy expenditure in the periphery. Analysis of mice with drug-induced sympathetic denervation suggested that these kinases controlled the sympathetic drive in the brain. Administration of inhibitors of both PI3Kβ and PI3Kγ to mice by intracerebroventricular delivery induced a 10% reduction in fat mass as quickly as 10 days. These results suggest that combined inhibition of PI3Kβ and PI3Kγ might represent a promising treatment for obesity.

The Gq signalling pathway inhibits brown and beige adipose tissue

Brown adipose tissue (BAT) dissipates nutritional energy as heat via the uncoupling protein-1 (UCP1) and BAT activity correlates with leanness in human adults. Here we profile G protein-coupled receptors (GPCRs) in brown adipocytes to identify druggable regulators of BAT. Twenty-one per cent of the GPCRs link to the Gq family, and inhibition of Gq signalling enhances differentiation of human and murine brown adipocytes. In contrast, activation of Gq signalling abrogates brown adipogenesis. We further identify the endothelin/Ednra pathway as an autocrine activator of Gq signalling in brown adipocytes. Expression of a constitutively active Gq protein in mice reduces UCP1 expression in BAT, whole-body energy expenditure and the number of brown-like/beige cells in white adipose tissue (WAT). Furthermore, expression of Gq in human WAT inversely correlates with UCP1 expression. Thus, our data indicate that Gq signalling regulates brown/beige adipocytes and inhibition of Gq signalling may be a novel therapeutic approach to combat obesity.

Regulation of metabolism by cGMP.

The second messenger cyclic guanosine monophosphate (cGMP) mediates the physiological effects of nitric oxide and natriuretic peptides in a broad spectrum of tissues and cells. So far, the major focus of research on cGMP lay on the cardiovascular system. Recent evidence suggests that cGMP also plays a major role in the regulation of cellular and whole-body metabolism. Here, we focus on the role of cGMP in adipose tissue. In addition, other organs important for the regulation of metabolism and their regulation by cGMP are discussed. Targeting the cGMP signaling pathway could be an exciting approach for the regulation of energy expenditure and the treatment of obesity.

A molecular mechanism for therapeutic effects of cGMP-elevating agents in pulmonary arterial hypertension

Background: Pulmonary arterial hypertension (PAH) is characterized by abnormal vascular remodeling and impaired BMP/Smad signaling. Results: cGMP/PKGI are required for the anti-proliferative and pro-differentiation effects of BMP in pulmonary artery smooth muscle cells (PASMCs) in vivo. Conclusion: Cooperative cGMP and BMP signaling is essential for maintaining a low proliferative, differentiated PASMC phenotype. Significance: These data explain therapeutic cGMP effects in PAH. Pulmonary arterial hypertension (PAH) is a progressive, usually fatal disease with abnormal vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients are hyperproliferative and apoptosis-resistant and demonstrate decreased signaling in response to bone morphogenetic proteins (BMPs). Cyclic GMP-elevating agents are beneficial in PAH, but their mechanism(s) of action are incompletely understood. Here we show that BMP signaling via Smad1/5/8 requires cGMP-dependent protein kinase isotype I (PKGI) to maintain PASMCs in a differentiated, low proliferative state. BMP cooperation with cGMP/PKGI was crucial for transcription of contractile genes and suppression of pro-proliferative and anti-apoptotic genes. Lungs from mice with low or absent PKGI (Prkg1+/− and Prkg1−/− mice) exhibited impaired BMP signaling, decreased contractile gene expression, and abnormal vascular remodeling. Conversely, cGMP stimulation of PKGI restored defective BMP signaling in rats with hypoxia-induced PAH, consistent with cGMP-elevating agents reversing vascular remodeling in this PAH model. Our results provide a mechanism for the therapeutic effects of cGMP-elevating agents in PAH and suggest that combining them with BMP mimetics may provide a novel, disease-modifying approach to PAH therapy.

Impaired border zone formation and adverse remodeling after reperfused myocardial infarction in cannabinoid CB2 receptor deficient mice

AIMS Reperfusion ofmyocardial infarction is associated with inflammatory reaction and subsequentmyocardial remodeling with a rapid scar formation in mice. The cannabinoid receptor CB2 has been associated with cardioprotection and regulation ofmacrophage function.Weinvestigated its role in remodeling of reperfused infarction. MAIN METHODS One hour LAD-occlusion was followed by reperfusion over 6 h and 1, 3 and 7 days in wild-type C57/BL6J (WT) and CB2 receptor-deficient (Cnr2−/−)mice (n=8/group). Hearts were processed for functional, morphological and mRNA/protein analysis, and tissue concentration of endocannabinoidswas determined using liquid chromatography-multiple reaction monitoring. KEY FINDINGS In contrast to a rapid formation of granulation tissue and a compacted non-transmural scar inWT mice after 7 days of reperfusion, Cnr2−/− mice showed a non-compacted transmural scar. Millar® left ventricular catheter measurements revealed a significantly worse function in Cnr2−/− mice.We found no compensatory elevation of endocannabinoid concentration in Cnr2−/− hearts. Macrophage infiltration was significantly stronger in Cnr2−/− hearts and affected also the remote septum, when compared to WT hearts.We found a cytokine-driven inflammatory response in Cnr2−/− hearts with no significant induction of chemokines. Immunohistochemistry for thrombospondin-1 revealed a dysfunctional infarction border zone formation in Cnr2−/− hearts. Cnr2−/−hearts showed no significant induction of tenascin C, collagen-Iα or lysil oxidase, thereby indicating adversemyocardial remodeling. SIGNIFICANCE Endocannabinoids act via CB2 receptor in the modulation of inflammatory response and myocardial remodeling after infarction. CB2 receptor plays an important role in the formation of infarction border zone, collagen deposition and organization of stable scar during remodeling.

Divergent effects of a designer natriuretic peptide CD-NP in the regulation of adipose tissue and metabolism

Objective Obesity is defined as an abnormal increase in white adipose tissue (WAT) and is a major risk factor for type 2 diabetes and cardiovascular disease. Brown adipose tissue (BAT) dissipates energy and correlates with leanness. Natriuretic peptides have been shown to be beneficial for brown adipocyte differentiation and browning of WAT. Methods Here, we investigated the effects of an optimized designer natriuretic peptide (CD-NP) on murine adipose tissues in vitro and in vivo. Results In murine brown and white adipocytes, CD-NP activated cGMP production, promoted adipogenesis, and increased thermogenic markers. Consequently, mice treated for 10 days with CD-NP exhibited increased “browning” of WAT. To study CD-NP effects on diet-induced obesity (DIO), we delivered CD-NP for 12 weeks. Although CD-NP reduced inflammation in WAT, CD-NP treated DIO mice exhibited a significant increase in body mass, worsened glucose tolerance, and hepatic steatosis. Long-term CD-NP treatment resulted in an increased expression of the NP scavenging receptor (NPR-C) and decreased lipolytic activity. Conclusions NP effects differed depending on the duration of treatment raising questions about the rational of natriuretic peptide treatment in obese patients.

Role of cGMP in fat and metabolism

Background The cGMP pathway regulates a large spectrum of physiological processes including metabolism. Our aim is to elucidate the cGMP signaling cascade in adipose tissue. Two types of fat tissue can be distinguished in mammals: white adipose tissue (WAT), which is the biggest storage of energy, and brown fat (BAT) that can dissipate energy as heat (non-shivering thermogenesis). We and others found that cGMP enhances differentiation of brown and white adipocytes [1-4].

Guanylyl cyclase-B plays an important role in adipogenesis and “browning” of white fat

Background Obesity, the excessive accumulation of adipose tissue, is the result of an imbalance in energy intake and output. Two functionally distinct types of adipose tissue are present in mammals: white adipose tissue (WAT) and brown adipose tissue (BAT). Beside its role as energy storage, WAT is also a metabolic active organ involved in inflammatory response, glucose metabolism and blood pressure by secreting adipokines. In contrast to WAT, BAT dissipates energy as heat. Previously, we have demonstrated essential role of the cGMP/PKGI in brown and white adipogenesis.