Linda S. Hoffmann

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

Nitric oxide-independent stimulation of soluble guanylate cyclase reduces organ damage in experimental low- renin and high-renin models

Objectives The nitric oxide–soluble guanylate cyclase (sGC)–cGMP signal transduction pathway is impaired in different cardiovascular diseases, including pulmonary hypertension, heart failure and arterial hypertension. Riociguat is a novel stimulator of soluble guanylate cyclase (sGC). However, little is known about the effects of sGC stimulators in experimental models of hypertension. We thus investigated the cardio-renal protective effects of riociguat in low-renin and high-renin rat models of hypertension. Methods The vasorelaxant effect of riociguat was tested in vitro on isolated saphenous artery rings of normal and nitrate tolerant rabbits. The cardiovascular in-vivo effects of sGC stimulation were evaluated in hypertensive renin-transgenic rats treated with the nitric oxide-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (high-renin model) and in rats with 5/6 nephrectomy (low-renin model). Results In both animal models, riociguat treatment improved survival and normalized blood pressure. Moreover, in the L-NAME study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight and lower cardiac interstitial fibrosis, and reduced renal target organ damage as indicated by lower plasma creatinine and urea, less glomerulosclerosis and less renal interstitial fibrosis. In the 5/6 nephrectomy study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight, lower myocyte diameter and lower arterial media/lumen ratio, and reduced renal target organ damage as indicated by improved creatinine clearance and less renal interstitial fibrosis. Conclusion We demonstrate for the first time that the novel sGC stimulator riociguat shows in two independent models of hypertension a potent protection against cardiac and renal target organ damage.

Brown, Beige, and White: The New Color Code of Fat and Its Pharmacological Implications

Brown adipose tissue (BAT) was previously regarded as a special type of fat relevant only for defending hibernating animals and newborns against a cold environment. Recently, BAT has received considerable attention following its (re)discovery in humans. Using glucose tracers, multiple laboratories independently found metabolically active BAT in adults. The enormous metabolic powers of BAT in animal models could make it an attractive target for antiobesity therapies in humans. Here, we review the present knowledge on the role of BAT in energy homeostasis and metabolism, focusing on signaling pathways and potential targets for novel therapeutics. We also shine light on ongoing debates, including those about the true color of brown fat in adults, as well as on the requirements for translation of basic research on BAT into clinical medicine.

Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGCs redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.

Filarial Infection or Antigen Administration Improves Glucose Tolerance in Diet-Induced Obese Mice.

Helminths induce type 2 immune responses and establish an anti-inflammatory milieu in their hosts. This immunomodulation was previously shown to improve diet-induced insulin resistance which is linked to chronic inflammation. In the current study, we demonstrate that infection with the filarial nematode Litomosoides sigmodontis increased the eosinophil number and alternatively activated macrophage abundance within epididymal adipose tissue (EAT) and improved glucose tolerance in diet-induced obese mice in an eosinophil-dependent manner. L. sigmodontis antigen (LsAg) administration neither altered the body weight of animals nor adipose tissue mass or adipocyte size, but it triggered type 2 immune responses, eosinophils, alternatively activated macrophages, and type 2 innate lymphoid cells in EAT. Improvement in glucose tolerance by LsAg treatment remained even in the absence of Foxp3+ regulatory T cells. Furthermore, PCR array results revealed that LsAg treatment reduced inflammatory immune responses and increased the expression of genes related to insulin signaling (Glut4, Pde3b, Pik3r1, and Hk2) and fatty acid uptake (Fabp4 and Lpl). Our investigation demonstrates that L. sigmodontis infection and LsAg administration reduce diet-induced EAT inflammation and improve glucose tolerance. Helminth-derived products may, therefore, offer new options to improve insulin sensitivity, while loss of helminth infections in developing and developed countries may contribute to the recent increase in the prevalence of type 2 diabetes.

Chronic Activation of Heme Free Guanylate Cyclase Leads to Renal Protection in Dahl Salt-Sensitive Rats

The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophasphate (cGMP)-signalling pathway is impaired under oxidative stress conditions due to oxidation and subsequent loss of the prosthetic sGC heme group as observed in particular in chronic renal failure. Thus, the pool of heme free sGC is increased under pathological conditions. sGC activators such as cinaciguat selectively activate the heme free form of sGC and target the disease associated enzyme. In this study, a therapeutic effect of long-term activation of heme free sGC by the sGC activator cinaciguat was investigated in an experimental model of salt-sensitive hypertension, a condition that is associated with increased oxidative stress, heme loss from sGC and development of chronic renal failure. For that purpose Dahl/ss rats, which develop severe hypertension upon high salt intake, were fed a high salt diet (8% NaCl) containing either placebo or cinaciguat for 21 weeks. Cinaciguat markedly improved survival and ameliorated the salt-induced increase in blood pressure upon treatment with cinaciguat compared to placebo. Renal function was significantly improved in the cinaciguat group compared to the placebo group as indicated by a significantly improved glomerular filtration rate and reduced urinary protein excretion. This was due to anti-fibrotic and anti-inflammatory effects of the cinaciguat treatment. Taken together, this is the first study showing that long-term activation of heme free sGC leads to renal protection in an experimental model of hypertension and chronic kidney disease. These results underline the promising potential of cinaciguat to treat renal diseases by targeting the disease associated heme free form of sGC.

Effects of obesity on sGCβ1 mediated signaling in white adipose tissue

Background Obesity has reached pandemic proportions with 1.9 billion overweight adults; 600 million of whom are obese [1]. It has been shown that stimulating cyclic 3’-5’ guanosine monophosphate (cGMP) signaling is able to promote adipose tissue homeostasis in obese mice [2]. Conversely, genetic depletion of cGMP dependent protein kinase 1 (PKG1), causes hepatic inflammation and fasting hyperglycemia in mice [3]. Furthermore, cGMP signaling is essential for differentiation of preadipocytes to mature adipocytes in vitro [4,5]. PKG1 activation blocks RhoA mediated inhibition of insulin receptor substrate 1 in adipocytes in vitro [4,5], thus maintaining insulin sensitivity. The status of cGMP signaling cascade in adipose tissue under obese conditions is not yet fully understood. Here, we examine the functional status of cGMP signaling in white adipose tissue (WAT) of lean and obese mice.

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].

Cardio-renal protection of riociguat (BAY 63-2521) in low- and high-renin models of hypertension

Address: 1Department of Nephrology, Charite, Campus Benjamin Franklin, Hindenburgdamm 30, Berlin, Germany, 2Center for Cardiovascular Research, Department of Pharmacology and Toxicology, Charite, Campus Mitte, Hessische Str. 3-4, Berlin, Germany, 3Pathology, Research Centre, Bayer HealthCare, Aprather Weg 18a, Wuppertal, Germany, 4Cardiology Research, Bayer HealthCare, Aprather Weg 18a, Wuppertal, Germany, 5Martin-Luther-University, School of Pharmacy, Halle, Germany and 6Department of Pharmacology, University of Cologne, Gleueler Strasse 24, D-50931 Cologne, Germany