Pál Pacher

Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity

Endogenous cannabinoids acting at CB(1) receptors stimulate appetite, and CB(1) antagonists show promise in the treatment of obesity. CB(1) (-/-) mice are resistant to diet-induced obesity even though their caloric intake is similar to that of wild-type mice, suggesting that endocannabinoids also regulate fat metabolism. Here, we investigated the possible role of endocannabinoids in the regulation of hepatic lipogenesis. Activation of CB(1) in mice increases the hepatic gene expression of the lipogenic transcription factor SREBP-1c and its targets acetyl-CoA carboxylase-1 and fatty acid synthase (FAS). Treatment with a CB(1) agonist also increases de novo fatty acid synthesis in the liver or in isolated hepatocytes, which express CB(1). High-fat diet increases hepatic levels of the endocannabinoid anandamide (arachidonoyl ethanolamide), CB(1) density, and basal rates of fatty acid synthesis, and the latter is reduced by CB(1) blockade. In the hypothalamus, where FAS inhibitors elicit anorexia, SREBP-1c and FAS expression are similarly affected by CB(1) ligands. We conclude that anandamide acting at hepatic CB(1) contributes to diet-induced obesity and that the FAS pathway may be a common molecular target for central appetitive and peripheral metabolic regulation.

Adenosine receptors: therapeutic aspects for inflammatory and immune diseases

Adenosine is a key endogenous molecule that regulates tissue function by activating four G-protein-coupled adenosine receptors: A1, A2A, A2B and A3. Cells of the immune system express these receptors and are responsive to the modulatory effects of adenosine in an inflammatory environment. Animal models of asthma, ischaemia, arthritis, sepsis, inflammatory bowel disease and wound healing have helped to elucidate the regulatory roles of the various adenosine receptors in dictating the development and progression of disease. This recent heightened awareness of the role of adenosine in the control of immune and inflammatory systems has generated excitement regarding the potential use of adenosine-receptor-based therapies in the treatment of infection, autoimmunity, ischaemia and degenerative diseases.

NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).

Measurement of cardiac function using pressure–volume conductance catheter technique in mice and rats

Ventricular pressure–volume relationships have become well established as the most rigorous and comprehensive ways to assess intact heart function. Thanks to advances in miniature sensor technology, this approach has been successfully translated to small rodents, allowing for detailed characterization of cardiovascular function in genetically engineered mice, testing effects of pharmacotherapies and studying disease conditions. This method is unique for providing measures of left ventricular (LV) performance that are more specific to the heart and less affected by vascular loading conditions. Here we present descriptions and movies for procedures employing this method (anesthesia, intubation and surgical techniques, calibrations). We also provide examples of hemodynamics measurements obtained from normal mice/rats, and from animals with cardiac hypertrophy/heart failure, and describe values for various useful load-dependent and load-independent indexes of LV function obtained using different types of anesthesia. The completion of the protocol takes 1–4 h (depending on the experimental design/end points).

Dysregulation of the Peripheral and Adipose Tissue Endocannabinoid System in Human Abdominal Obesity

The endocannabinoid system has been suspected to contribute to the association of visceral fat accumulation with metabolic diseases. We determined whether circulating endocannabinoids are related to visceral adipose tissue mass in lean, subcutaneous obese, and visceral obese subjects (10 men and 10 women in each group). We further measured expression of the cannabinoid type 1 (CB1) receptor and fatty acid amide hydrolase (FAAH) genes in paired samples of subcutaneous and visceral adipose tissue in all 60 subjects. Circulating 2-arachidonoyl glycerol (2-AG) was significantly correlated with body fat (r = 0.45, P = 0.03), visceral fat mass (r = 0.44, P = 0.003), and fasting plasma insulin concentrations (r = 0.41, P = 0.001) but negatively correlated to glucose infusion rate during clamp (r = 0.39, P = 0.009). In visceral adipose tissue, CB1 mRNA expression was negatively correlated with visceral fat mass (r = 0.32, P = 0.01), fasting insulin (r = 0.48, P < 0.001), and circulating 2-AG (r = 0.5, P < 0.001), whereas FAAH gene expression was negatively correlated with visceral fat mass (r = 0.39, P = 0.01) and circulating 2-AG (r = 0.77, P < 0.001). Our findings suggest that abdominal fat accumulation is a critical correlate of the dysregulation of the peripheral endocannabinoid system in human obesity. Thus, the endocannabinoid system may represent a primary target for the treatment of abdominal obesity and associated metabolic changes.

Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

CD39 and CD73 in immunity and inflammation

The enzymatic activities of CD39 and CD73 play strategic roles in calibrating the duration, magnitude, and chemical nature of purinergic signals delivered to immune cells through the conversion of ADP/ATP to AMP and AMP to adenosine, respectively. This drives a shift from an ATP-driven proinflammatory environment to an anti-inflammatory milieu induced by adenosine. The CD39/CD73 pathway changes dynamically with the pathophysiological context in which it is embedded. It is becoming increasingly appreciated that altering this catabolic machinery can change the course or dictate the outcome of several pathophysiological events, such as AIDS, autoimmune diseases, infections, atherosclerosis, ischemia-reperfusion injury, and cancer, suggesting these ectoenzymes are novel therapeutic targets for managing a variety of disorders.

Soluble guanylate cyclase as an emerging therapeutic target in cardiopulmonary disease.

Soluble guanylate cyclase (sGC), a key enzyme of the nitric oxide (NO) signaling pathway, is attracting rapidly growing interest as a therapeutic target in cardiopulmonary disease, with several sGC agonists currently in clinical development. On binding of NO to a prosthetic heme group on sGC, the enzyme catalyzes synthesis of the second messenger cGMP, which produces vasorelaxation and inhibits smooth muscle proliferation, leukocyte recruitment, and platelet aggregation through a number of downstream mechanisms.1,2 Impaired NO and cGMP signaling has been implicated in the pathogenesis of cardiovascular disease, including systemic arterial and pulmonary hypertension (PH), coronary artery disease, peripheral vascular disease (including erectile dysfunction), and atherosclerosis.1,3,–,5 Organic nitrates that target the NO signaling pathway have been used to treat cardiovascular disease for >150 years. More recently, gaseous NO administered by inhalation has been approved for the treatment of persistent PH of the newborn.3,6 These agents nonetheless have several important limitations. Cardiovascular disease is associated with resistance to NO and organic nitrates.7 This may be due to the oxidative stress–induced alteration of the redox state of the prosthetic heme on sGC (from ferrous to ferric) that weakens the binding of heme to the enzyme and renders sGC unresponsive to NO.1,8 Furthermore, the long-term efficacy of organic nitrates is limited by the development of tolerance.9 Nitric oxide may also have numerous cytotoxic effects, mostly attributed to the reactive oxidant peroxynitrite (formed from the diffusion-controlled reaction of NO with superoxide).3,10 Peroxynitrite interacts with proteins and lipids, altering cellular signaling, disrupting mitochondrial function, and damaging DNA, which can eventually culminate in cellular dysfunction and/or death.3 Because the beneficial effects of NO appear to be …

Resveratrol induces mitochondrial biogenesis in endothelial cells

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Role of the Peroxynitrite-Poly(ADP-Ribose) Polymerase Pathway in Human Disease

Throughout the last 2 decades, experimental evidence from in vitro studies and preclinical models of disease has demonstrated that reactive oxygen and nitrogen species, including the reactive oxidant peroxynitrite, are generated in parenchymal, endothelial, and infiltrating inflammatory cells during stroke, myocardial and other forms of reperfusion injury, myocardial hypertrophy and heart failure, cardiomyopathies, circulatory shock, cardiovascular aging, atherosclerosis and vascular remodeling after injury, diabetic complications, and neurodegenerative disorders. Peroxynitrite and other reactive species induce oxidative DNA damage and consequent activation of the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1), the most abundant isoform of the PARP enzyme family. PARP overactivation depletes its substrate NAD(+), slowing the rate of glycolysis, electron transport, and ATP formation, eventually leading to functional impairment or death of cells, as well as up-regulation of various proinflammatory pathways. In related animal models of disease, peroxynitrite neutralization or pharmacological inhibition of PARP provides significant therapeutic benefits. Therefore, novel antioxidants and PARP inhibitors have entered clinical development for the experimental therapy of various cardiovascular and other diseases. This review focuses on the human data available on the pathophysiological relevance of the peroxynitrite-PARP pathway in a wide range of disparate diseases, ranging from myocardial ischemia/reperfusion injury, myocarditis, heart failure, circulatory shock, and diabetic complications to atherosclerosis, arthritis, colitis, and neurodegenerative disorders.