PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism.

Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK31-4. Environmental factors can explain differences in the natural history of CCMs between individuals5, but why single CCMs often exhibit sudden, rapid growth, culminating in strokes or seizures, is unknown. Here we show that growth of CCMs requires increased signalling through the phosphatidylinositol-3-kinase (PI3K)-mTOR pathway as well as loss of function of the CCM complex. We identify somatic gain-of-function mutations in PIK3CA and loss-of-function mutations in the CCM complex in the same cells in a majority of human CCMs. Using mouse models, we show that growth of CCMs requires both PI3K gain of function and CCM loss of function in endothelial cells, and that both CCM loss of function and increased expression of the transcription factor KLF4 (a downstream effector of MEKK3) augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor rapamycin effectively blocks the formation of CCMs in mouse models. We establish a three-hit mechanism analogous to cancer, in which aggressive vascular malformations arise through the loss of vascular 'suppressor genes' that constrain vessel growth and gain of a vascular 'oncogene' that stimulates excess vessel growth. These findings suggest that aggressive CCMs could be treated using clinically approved mTORC1 inhibitors.

In atrial fibrillation epilepsy risk differs between oral anticoagulants: active comparator, nested case-control study.

AIMS: Atrial fibrillation (AF) is a risk factor for brain infarction, which can lead to epilepsy. We aimed to investigate whether treatment of AF with direct oral anticoagulants (DOACs) affects the risk of epilepsy in comparison to treatment with the vitamin K antagonist phenprocoumon (PPC). METHODS AND RESULTS: We performed an active comparator, nested case-control study based on the German Pharmacoepidemiological Research Database that includes claims data from statutory health insurance providers of about 25 million persons since 2004. In 2011-17, 227 707 AF patients initiated treatment with a DOAC or PPC, of which 1828 cases developed epilepsy on current treatment with an oral anticoagulant. They were matched to 19 084 controls without epilepsy. Patients with DOAC treatment for AF had an overall higher risk of epilepsy with an odds ratio of 1.39, 95% CI (1.24; 1.55) compared to current PPC treatment. Cases had higher baseline CHA2DS2-VASc scores and more frequently a history of stroke than controls. After excluding patients with ischaemic stroke prior to the diagnosis of epilepsy, the risk of epilepsy was still higher on DOACs than on PPC. In contrast, within a cohort of patients with venous thromboembolism, the risk of epilepsy on treatment with DOACs was less elevated [adjusted odds ratio 1.15, 95% CI (0.98; 1.34)]. CONCLUSION: In patients with AF initiating oral anticoagulation, treatment with a DOAC was associated with an increased risk of epilepsy compared to the vitamin K antagonist PPC. Covert brain infarction may explain the observed elevated risk of epilepsy.

Gene therapy targeting the blood-brain barrier improves neurological symptoms in a model of genetic MCT8 deficiency.

A genetic deficiency of the solute carrier monocarboxylate transporter 8 (MCT8), termed Allan-Herndon-Dudley syndrome, is an important cause of X-linked intellectual and motor disability. MCT8 transports thyroid hormones across cell membranes. While thyroid hormone analogues improve peripheral changes of MCT8 deficiency, no treatment of the neurological symptoms is available so far. Therefore, we tested a gene replacement therapy in Mct8- and Oatp1c1-deficient mice as a well-established model of the disease. Here, we report that targeting brain endothelial cells for Mct8 expression by intravenously injecting the vector AAV-BR1-Mct8 increased tri-iodothyronine (T3) levels in the brain and ameliorated morphological and functional parameters associated with the disease. Importantly, the therapy resulted in a long-lasting improvement in motor coordination. Thus, the data support the concept that MCT8 mediates the transport of thyroid hormones into the brain and indicate that a readily accessible vascular target can help overcome the consequences of the severe disability associated with MCT8 deficiency.

Impaired endothelium-mediated cerebrovascular reactivity promotes anxiety and respiration disorders in mice.

Carbon dioxide (CO2), the major product of metabolism, has a strong impact on cerebral blood vessels, a phenomenon known as cerebrovascular reactivity. Several vascular risk factors such as hypertension or diabetes dampen this response, making cerebrovascular reactivity a useful diagnostic marker for incipient vascular pathology, but its functional relevance, if any, is still unclear. Here, we found that GPR4, an endothelial H+ receptor, and endothelial Gαq/11 proteins mediate the CO2/H+ effect on cerebrovascular reactivity in mice. CO2/H+ leads to constriction of vessels in the brainstem area that controls respiration. The consequential washout of CO2, if cerebrovascular reactivity is impaired, reduces respiration. In contrast, CO2 dilates vessels in other brain areas such as the amygdala. Hence, an impaired cerebrovascular reactivity amplifies the CO2 effect on anxiety. Even at atmospheric CO2 concentrations, impaired cerebrovascular reactivity caused longer apneic episodes and more anxiety, indicating that cerebrovascular reactivity is essential for normal brain function. The site-specific reactivity of vessels to CO2 is reflected by regional differences in their gene expression and the release of vasoactive factors from endothelial cells. Our data suggest the central nervous system (CNS) endothelium as a target to treat respiratory and affective disorders associated with vascular diseases.

Is LRP2 Involved in Leptin Transport over the Blood-Brain Barrier and Development of Obesity?

The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.

Regulation of Thyroid Hormone Gatekeepers by Thyrotropin in Tanycytes.

Background: Tanycytes are specialized glial cells within the mediobasal hypothalamus that have multiple functions, including hormone sensing and regulation of hypophysiotropic hormone secretion. There are ongoing discussions about the role of tanycytes in regulating the supply of hypothalamic thyroid hormones (THs) through the expression of TH transporters (Slc16a2, Slco1c1) and deiodinases (Dio2, Dio3). In this study, we investigated the potential feedback effect of thyrotropin (TSH) on the transcription of these gatekeeper genes on tanycytes. Methods: We analyzed the changes in the expression of TH-gatekeeper genes, in TSH-stimulated primary tanycytes, using quantitative polymerase chain reaction (qPCR). We also used RNAScope® in brain slices to further reveal the local distribution of the transcripts. In addition, we blocked intracellular pathways and used small-interfering RNA (siRNA) to elucidate differences in the regulation of the gatekeeper genes. Results: TSH elevated messenger RNA (mRNA) levels of Slco1c1, Dio2, and Dio3 in tanycytes, while Slc16a2 was mostly unaffected. Blockade and knockdown of the TSH receptor (TSHR) and antagonization of cAMP response element-binding protein (CREB) clearly abolished the increased expression induced by TSH, indicating PKA-dependent regulation through the TSHR. The TSH-dependent expression of Dio3 and Slco1c1 was also regulated by protein kinase C (PKC), and in case of Dio3, also by extracellular signal-regulated kinase (ERK) activity. Importantly, these gene regulations were specifically found in different subpopulations of tanycytes. Conclusions: This study demonstrates that TSH induces transcriptional regulation of TH-gatekeeper genes in tanycytes through the Tshr/Gαq/PKC pathway, in parallel to the Tshr/Gαs/PKA/CREB pathway. These differential actions of TSH on tanycytic subpopulations appear to be important for coordinating the supply of TH to the hypothalamus and aid its functions.

Partial Resistance to Thyroid Hormone-Induced Tachycardia and Cardiac Hypertrophy in Mice Lacking Thyroid Hormone Receptor ß.

Background: Thyroid hormones regulate cardiac functions mainly through direct actions in the heart and by binding to the thyroid hormone receptor (TR) isoforms α1 and ß. While the role of the most abundantly expressed isoform, TRα1, is widely studied and well characterized, the role of TRß in regulating heart functions is still poorly understood, primarily due to the accompanying elevation of circulating thyroid hormone in TRß knockout mice (TRß-KO). However, their hyperthyroidism is ameliorated at thermoneutrality, which allows studying the role of TRß without this confounding factor. Methods: Here, we noninvasively monitored heart rate in TRß-KO mice over several days using radiotelemetry at different housing temperatures (22°C and 30°C) and upon 3,3',5-triiodothyronine (T3) administration in comparison to wild-type animals. Results: TRß-KO mice displayed normal average heart rate at both 22°C and 30°C with only minor changes in heart rate frequency distribution, which was confirmed by independent electrocardiogram recordings in freely-moving conscious mice. Parasympathetic nerve activity was, however, impaired in TRß-KO mice at 22°C, and only partly rescued at 30°C. As expected, oral treatment with pharmacological doses of T3 at 30°C led to tachycardia in wild-types, accompanied by broader heart rate frequency distribution and increased heart weight. The TRß-KO mice, in contrast, showed blunted tachycardia, as well as resistance to changes in heart rate frequency distribution and heart weight. At the molecular level, these observations were paralleled by a blunted cardiac mRNA induction of several important genes, including the pacemaker channels Hcn2 and Hcn4, as well as Kcna7. Conclusions: The phenotyping of TRß-KO mice conducted at thermoneutrality allows novel insights on the role of TRß in cardiac functions in the absence of the usual confounding hyperthyroidism. Even though TRß is expressed at lower levels than TRα1 in the heart, our findings demonstrate an important role for this isoform in the cardiac response to thyroid hormones.

High-fat diet-induced hyperinsulinemia and tissue-specific insulin resistance in Cry-deficient mice.

Perturbation of circadian rhythmicity in mammals, either by environmental influences such as shiftwork or by genetic manipulation, has been associated with metabolic disturbance and the development of obesity and diabetes. Circadian clocks are based on transcriptional/translational feedback loops, comprising positive and negative components. Whereas the metabolic effects of deletion of the positive arm of the clock gene machinery, as in Clock- or Bmal1-deficient mice, have been well characterized, inactivation of Period genes (Per1-3) as components of the negative arm have more complex, sometimes contradictory effects on energy homeostasis. The CRYPTOCHROMEs are critical interaction partners of PERs, and simultaneous deletion of Cry1 and -2 results in behavioral and molecular circadian arrhythmicity. We show that, when challenged with a high-fat diet, Cry1/2(-/-) mice rapidly gain weight and surpass that of wild-type mice, despite displaying hypophagia. Transcript analysis of white adipose tissue reveals upregulated expression of lipogenic genes, many of which are insulin targets. High-fat diet-induced hyperinsulinemia, as a result of potentiated insulin secretion, coupled with selective insulin sensitivity in adipose tissue of Cry1/2(-/-) mice, correlates with increased lipid uptake. Collectively, these data indicate that Cry deficiency results in an increased vulnerability to high-fat diet-induced obesity that might be mediated by increased insulin secretion and lipid storage in adipose tissues.

Short regulatory DNA sequences to target brain endothelial cells for gene therapy.

Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

The SARS-CoV-2 main protease Mpro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

The Role of Tanycytes in the Hypothalamus-Pituitary-Thyroid Axis and the Possibilities for Their Genetic Manipulation.

Thyroid hormone (TH) regulation is important for development, energy homeostasis, heart function, and bone formation. To control the effects of TH in target organs, the hypothalamus-pituitary-thyroid (HPT) axis and the tissue-specific availability of TH are highly regulated by negative feedback. To exert a central feedback, TH must enter the brain via specific transport mechanisms and cross the blood-brain barrier. Here, tanycytes, which are located in the ventral walls of the 3rd ventricle in the mediobasal hypothalamus (MBH), function as gatekeepers. Tanycytes are able to transport, sense, and modify the release of hormones of the HPT axis and are involved in feedback regulation. In this review, we focus on the relevance of tanycytes in thyrotropin-releasing hormone (TRH) release and review available genetic tools to investigate the physiological functions of these cells.

MCH Neurons Regulate Permeability of the Median Eminence Barrier.

Melanin-concentrating hormone (MCH)-expressing neurons are key regulators of energy and glucose homeostasis. Here, we demonstrate that they provide dense projections to the median eminence (ME) in close proximity to tanycytes and fenestrated vessels. Chemogenetic activation of MCH neurons as well as optogenetic stimulation of their projections in the ME enhance permeability of the ME by increasing fenestrated vascular loops and enhance leptin action in the arcuate nucleus of the hypothalamus (ARC). Unbiased phosphoRiboTrap-based assessment of cell activation upon chemogenetic MCH neuron activation reveals MCH-neuron-dependent regulation of endothelial cells. MCH neurons express the vascular endothelial growth factor A (VEGFA), and blocking VEGF-R signaling attenuates the leptin-sensitizing effect of MCH neuron activation. Our experiments reveal that MCH neurons directly regulate permeability of the ME barrier, linking the activity of energy state and sleep regulatory neurons to the regulation of hormone accessibility to the ARC.

NF-κB signaling in tanycytes mediates inflammation-induced anorexia.

OBJECTIVES: Infections, cancer, and systemic inflammation elicit anorexia. Despite the medical significance of this phenomenon, the question of how peripheral inflammatory mediators affect the central regulation of food intake is incompletely understood. Therefore, we have investigated the sickness behavior induced by the prototypical inflammatory mediator IL-1ß. METHODS: IL-1ß was injected intravenously. To interfere with IL-1ß signaling, we deleted the essential modulator of NF-κB signaling (Nemo) in astrocytes and tanycytes. RESULTS: Systemic IL-1ß increased the activity of the transcription factor NF-κB in tanycytes of the mediobasal hypothalamus (MBH). By activating NF-κB signaling, IL-1ß induced the expression of cyclooxygenase-2 (Cox-2) and stimulated the release of the anorexigenic prostaglandin E2 (PGE2) from tanycytes. When we deleted Nemo in astrocytes and tanycytes, the IL-1ß-induced anorexia was alleviated whereas the fever response and lethargy response were unchanged. Similar results were obtained after the selective deletion of Nemo exclusively in tanycytes. CONCLUSIONS: Tanycytes form the brain barrier that mediates the anorexic effect of systemic inflammation in the hypothalamus.

Nesfatin-1 decreases the motivational and rewarding value of food.

Homeostatic and hedonic pathways distinctly interact to control food intake. Dysregulations of circuitries controlling hedonic feeding may disrupt homeostatic mechanisms and lead to eating disorders. The anorexigenic peptides nucleobindin-2 (NUCB2)/nesfatin-1 may be involved in the interaction of these pathways. The endogenous levels of this peptide are regulated by the feeding state, with reduced levels following fasting and normalized by refeeding. The fasting state is associated with biochemical and behavioral adaptations ultimately leading to enhanced sensitization of reward circuitries towards food reward. Although NUCB2/nesfatin-1 is expressed in reward-related brain areas, its role in regulating motivation and preference for nutrients has not yet been investigated. We here report that both dopamine and GABA neurons express NUCB2/nesfatin-1 in the VTA. Ex vivo electrophysiological recordings show that nesfatin-1 hyperpolarizes dopamine, but not GABA, neurons of the VTA by inducing an outward potassium current. In vivo, central administration of nesfatin-1 reduces motivation for food reward in a high-effort condition, sucrose intake and preference. We next adopted a 2-bottle choice procedure, whereby the reward value of sucrose was compared with that of a reference stimulus (sucralose + optogenetic stimulation of VTA dopamine neurons) and found that nesfatin-1 fully abolishes the fasting-induced increase in the reward value of sucrose. These findings indicate that nesfatin-1 reduces energy intake by negatively modulating dopaminergic neuron activity and, in turn, hedonic aspects of food intake. Since nesfatin-1´s actions are preserved in conditions of leptin resistance, the present findings render the NUCB2/nesfatin-1 system an appealing target for the development of novel therapeutical treatments towards obesity.

The LepR-mediated leptin transport across brain barriers controls food reward.

OBJECTIVE: Leptin is a key hormone in the control of appetite and body weight. Predominantly produced by white adipose tissue, it acts on the brain to inhibit homeostatic feeding and food reward. Leptin has free access to circumventricular organs, such as the median eminence, but entry into other brain centers is restricted by the blood-brain and blood-CSF barriers. So far, it is unknown for which of its central effects leptin has to penetrate brain barriers. In addition, the mechanisms mediating the transport across barriers are unclear although high expression in brain barriers suggests an important role of the leptin receptor (LepR). METHODS: We selectively deleted LepR in brain endothelial and epithelial cells of mice (LepRbeKO). The expression of LepR in fenestrated vessels of the periphery and the median eminence as well as in tanycytes was not affected. RESULTS: Perfusion studies showed that leptin uptake by the brain depended on LepR in brain barriers. When being fed with a rewarding high-fat diet LepRbeKO mice gained more body weight than controls. The aggravated obesity of LepRbeKO mice was due to hyperphagia and a higher sensitivity to food reward. CONCLUSIONS: The LepR-mediated transport of leptin across brain barriers in endothelial cells lining microvessels and in epithelial cells of the choroid plexus controls food reward but is apparently not involved in homeostatic control of feeding.

Obesity-stimulated aldosterone release is not related to an S1P-dependent mechanism.

Aldosterone has been identified as an important factor in obesity-associated hypertension. Here, we investigated whether sphingosine-1-phosphate (S1P), which has previously been linked to obesity, increases aldosterone release. S1P-induced aldosterone release was determined in NCI H295R cells in the presence of S1P receptor (S1PR) antagonists. In vivo release of S1P (100-300 µg/kgbw) was investigated in pithed, lean Sprague Dawley (SD) rats, diet-obese spontaneous hypertensive rats (SHRs), as well as in lean or obese Zucker rats. Aldosterone secretion was increased in NCI H295R cells by S1P, the selective S1PR1 agonist SEW2871 and the selective S1PR2 antagonist JTE013. Treatment with the S1PR1 antagonist W146 or fingolimod and the S1PR1/3 antagonist VPbib2319 decreased baseline and/or S1P-stimulated aldosterone release. Compared to saline-treated SD rats, plasma aldosterone increased by ~50 pg/mL after infusing S1P. Baseline levels of S1P and aldosterone were higher in obese than in lean SHRs. Adrenal S1PR expression did not differ between chow- or CD-fed rats that had the highest S1PR1 and lowest S1PR4 levels. S1P induced a short-lasting increase in plasma aldosterone in obese, but not in lean SHRs. However, 2-ANOVA did not demonstrate any difference between lean and obese rats. S1P-induced aldosterone release was also similar between obese and lean Zucker rats. We conclude that S1P is a local regulator of aldosterone production. S1PR1 agonism induces an increase in aldosterone secretion, while stimulating adrenal S1PR2 receptor suppresses aldosterone production. A significant role of S1P in influencing aldosterone secretion in states of obesity seems unlikely.

Glucagon increase after chronic AT1 blockade is more likely related to an indirect leptin-dependent than to a pancreatic α-cell-dependent mechanism.

AT1 blockers (ARB) prevent diabetes by improving pancreatic ß cell function. Less is known about whether α cells are affected although they express angiotensin II (AngII) receptors. We aimed to investigate glucagon release upon AngII stimulation. We determined glucagon release after AngII stimulation (0.01-100 µM) in α cells (InR1G9) and isolated murine islets. We determined plasma glucagon in rats that were chronically treated with AngII (9 µg/h) or the ARBs telmisartan (8 mg/kg/day) and candesartan (16 mg/kg/day) and correlated glucagon with additional hormones (e.g. leptin). Glucagon was only released from InR1G9 cells and islets at the highest AngII concentrations (>10 µM). This was not inhibited by losartan or PD123319. Ang(1-7) and AngIV were also almost ineffective. AngII did not alter glucagon secretion from islets. Plasma glucagon increased when obese Zucker rats were treated with AngII or candesartan and also when Sprague Dawley rats were treated with telmisartan in parallel to high-calorie feeding. Plasma glucagon and leptin negatively correlated in ARB-treated rats. The glucagon release from InR1G9 cells or islets after AngII, AngIV or Ang(1-7) is unspecific since it only occurs, if at all, after the highest concentrations and cannot be blocked by specific inhibitors. Thus, the AngII-dependent increase in plasma glucagon seems to be mediated by indirect mechanisms. The negative correlations between plasma leptin and glucagon confirm findings showing that leptin suppresses glucagon release, leading us to suppose that the increase in plasma glucagon is related to the decrease in leptin after ARB treatment.

Tanycytes control the hormonal output of the hypothalamic-pituitary-thyroid axis.

The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating thyroid hormone levels in a narrow physiological range. As axons containing thyrotropin-releasing hormone (TRH) terminate on hypothalamic tanycytes, these specialized glial cells have been suggested to influence the activity of the HPT axis, but their exact role remained enigmatic. Here, we demonstrate that stimulation of the TRH receptor 1 increases intracellular calcium in tanycytes of the median eminence via Gαq/11 proteins. Activation of Gαq/11 pathways increases the size of tanycyte endfeet that shield pituitary vessels and induces the activity of the TRH-degrading ectoenzyme. Both mechanisms may limit the TRH release to the pituitary. Indeed, blocking TRH signaling in tanycytes by deleting Gαq/11 proteins in vivo enhances the response of the HPT axis to the chemogenetic activation of TRH neurons. In conclusion, we identify new TRH- and Gαq/11-dependent mechanisms in the median eminence by which tanycytes control the activity of the HPT axis.The hypothalamic-pituitary-thyroid (HPT) axis regulates a wide range of physiological processes. Here the authors show that hypothalamic tanycytes play a role in the homeostatic regulation of the HPT axis; activation of TRH signaling in tanycytes elevates their intracellular Ca2+ via Gαq/11 pathway, ultimately resulting in reduced TRH release into the pituitary vessels.

Preventing leptin resistance by blocking angiotensin II AT1 receptors in diet-induced obese rats.

BACKGROUND AND PURPOSE: AT1 receptor blockers (ARBs) represent an approach for treating metabolic syndrome due to their potency in reducing hypertension, body weight and onset of type 2 diabetes. The mechanism underlying ARB-induced weight loss is still unclear. EXPERIMENTAL APPROACH: Leptin resistance tests (LRTs) in diet-induced obese or lean rats were conducted to determine whether telmisartan (8 mg·kg(-1) ·day(-1) , 14 days) enhances leptin sensitivity. Phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) staining was performed in hypothalami to determine leptin transport across the blood-brain barrier. KEY RESULTS: Telmisartin reduced weight gain, food intake and plasma leptin but blood pressure remained unchanged. The 24 h profiles of plasma leptin after saline injections were similar in controls and telmisartan-treated rats, but after leptin injections were higher in controls and slightly lower in telmisartan-treated animals. After telmisartan, energy intake during LRT was lower in leptin- than in saline-pretreated rats, but remained unchanged in controls, irrespectively of whether rats received saline or leptin. Leptin minimized the gain in body weight during LRT in telmisartan-treated rats as compared with saline-treated animals. pSTAT3 staining was reduced in cafeteria diet-fed rats as compared with chow-fed rats but this was normalized by telmisartan. Telmisartin reduced hypothalamic mRNA levels of the orexigenic peptides melanin-concentrating hormone and prepro-orexin. CONCLUSIONS AND IMPLICATIONS: Rats fed a cafeteria diet develop leptin resistance after 2 weeks. Leptin sensitivity was preserved by telmisartan treatment even in rats fed a cafeteria diet. This pleiotropic effect is not related to the hypotensive action of telmisartan.

Zanamivir Amidoxime- and N-Hydroxyguanidine-Based Prodrug Approaches to Tackle Poor Oral Bioavailability.

The neuraminidase (NA) inhibitor zanamivir (1) is potently active against a broad panel of influenza A and B strains, including mutant viruses, but suffers from pharmacokinetic (PK) shortcomings. Here, distinct prodrug approaches are described that aimed at overcoming zanamivir's lack of oral bioavailability. Lowering the high basicity of the 4-guanidino group in zanamivir and of a bioisosteric 4-acetamidine analog (5) by N-hydroxylation was deemed to be a plausible tactic. The carboxylic acid and glycerol side chain were also masked with different ester groups. The bioisosteric amidine 5 turned out to be potently active against a panel of H1N1 (IC50 = 2-10 nM) and H3N2 (IC50 = 5-10 nM) influenza A viruses (NA inhibition assay). In vitro PK studies showed that all prodrugs were highly soluble, exhibited low protein binding, and were bioactivated by N-reduction to the respective guanidines and amidines. The most promising prodrug candidates, amidoxime ester 7 and N-hydroxyguanidine ester 8, were subjected to in vivo bioavailability studies. Unfortunately, both prodrugs were not orally bioavailable to a convincing degree (F ≤ 3.7%, rats). This finding questions the general feasibility of improving the oral bioavailability of 1 by lipophilicity-increasing prodrug strategies, and suggests that intrinsic structural features represent key hurdles.