Characterization of Micro-RNA Changes during the Progression of Type 2 Diabetes in Zucker Diabetic Fatty Rats.

The aim of the present pilot study was the identification of micro-RNA changes over time during the development and progression of type 2 diabetes (T2D) in Zucker diabetic fatty rats (ZDF rats). T2D is a complex metabolic disorder that is characterized, inter alia, by progressive failure of pancreatic ß cells to produce insulin, but also by functional or morphological modifications of others organ, such as liver, adipose tissue and the cardiovascular system. Micro-RNAs are a novel class of biomarkers that have the potential to represent biomarkers of disease progression. In this study, the onset and progression of diabetes was followed in ZDF rats from six weeks until 17 weeks of age. After an initial phase of hyperinsulinemia, the animals developed T2D and lost the capacity to produce sufficient insulin. Circulating miRNAs were measured from plasma samples at four time points: pre-diabetes (six weeks of age), hyperinsulinemia (eight weeks), ß cell failure (11 weeks) and late-stage diabetes (17 weeks) using TaqMan miRNA arrays. Bioinformatic analysis revealed distinct changes of circulating miRNAs over time. Several miRNAs were found to be increased over the course of the disease progression, such as miR-122, miR-133, miR-210 and miR-375. The most significantly decreased miRNAs were miR-140, miR-151-3p, miR-185, miR-203, miR-434-3p and miR-450a. Some of the miRNAs have also been identified in type 2 diabetic patients recently and, therefore, may have the potential to be useful biomarkers for the disease progression of T2D and/or the treatment response for anti-diabetic medications.

Effects of a single dose of amisulpride on functional brain changes during reward- and motivation-related processing using task-based fMRI in healthy subjects and patients with major depressive disorder - study protocol for a randomized clinical trial.

BACKGROUND: Anhedonia and other deficits in reward- and motivation-related processing in psychiatric patients, including patients with major depressive disorder (MDD), represent a high unmet medical need. Neurobiologically, these deficits in MDD patients are mainly associated with low dopamine function in a frontostriatal network. In this study, alterations in brain activation changes during reward processing and at rest in MDD patients compared with healthy subjects are explored and the effects of a single low dose of the dopamine D2 receptor antagonist amisulpride are investigated. METHODS: This is a randomized, controlled, double-blind, single-dose, single-center parallel-group clinical trial to assess the effects of a single dose of amisulpride (100 mg) on blood-oxygenation-level-dependent (BOLD) responses during reward- and motivation-related processing in healthy subjects (n = 60) and MDD patients (n = 60). Using functional magnetic resonance imaging (fMRI), BOLD responses are assessed during the monetary incentive delay (MID) task (primary outcome). Exploratory outcomes include BOLD responses and behavioral measures during the MID task, instrumental learning task, effort-based decision-making task, social incentive delay task, and probabilistic reward task as well as changes in resting state functional connectivity and cerebral blood flow. DISCUSSION: This study broadly covers all aspects of reward- and motivation-related processing as categorized by the National Institute of Mental Health Research Domain Criteria and is thereby an important step towards precision psychiatry. Results regarding the immediate effects of a dopaminergic drug on deficits in reward- and motivation-related processing not only have the potential to significantly broaden our understanding of underlying neurobiological processes but might eventually also pave the way for new treatment options. TRIAL REGISTRATION: ClinicalTrials.gov NCT05347199. April 12, 2022.

The glucose-dependent insulinotropic polypeptide (GIP) regulates body weight and food intake via CNS-GIPR signaling.

Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.

Differences in kidney-specific DPP-4 inhibition by linagliptin and sitagliptin.

The two dipeptidyl peptidase (DPP)-4 inhibitors, linagliptin and sitagliptin, were shown to exert different binding kinetics in vitro. Twenty-four hours after oral dosing particularly in vivo inhibition of renal-specific DPP-4 activity was more sustained in Sprague Dawley rats after exposure to linagliptin than it was after sitagliptin.

Discovery and translation of a target engagement marker for AMP-activated protein kinase (AMPK).

BACKGROUND: Activation of the AMP-activated protein kinase (AMPK) is an attractive approach for the treatment of type 2 diabetes. AMPK activation reduces glucose levels in animal models of type 2 diabetes by increasing glucose uptake in skeletal muscles and reducing hepatic glucose production. Furthermore, AMPK activation ameliorates hepatic steatosis in animal models. For the clinical development of AMPK activators it is essential to have a reliable target engagement marker for appropriate dose finding and to support proof of clinical principle. While the activation of AMPK by quantification of the phosphorylation of AMPK at Thr172 in target tissues can be assessed pre-clinically, this is not feasible in clinical studies. Therefore, we attempted to identify and translate a peripheral target engagement biomarker downstream of AMPK activation for clinical use in blood samples. METHODS: For pharmacological activation of AMPK, two AMPK activators were synthesized (compound 1 and 2). A compound with structural similarities but no pharmacological effect on AMPK phosphorylation was synthesized as negative control (compound 3). Whole blood from healthy volunteers was incubated with an AMPK activator for up to 6 hours and mRNA sequencing was performed. Additionally, human PBMCs were isolated to evaluate Thr172-phosphorylation of AMPK in Western blots. In order to enable identification of translatable biomarker candidates, blood samples from HanWistar rats treated for two weeks with an AMPK activator were also subjected to mRNA sequencing. Furthermore, concentration-response curves for four biomarker candidates were recorded in human blood samples using Nanostring nCounter technology. Finally, ZDF rats were treated with increasing doses of compound 2 for five weeks to investigate the glucose-lowering efficacy. To investigate changes of mRNA expression of two selected biomarker candidates in this ZDF rat study, qRT-PCR was performed. RESULTS: Pharmacological activation of AMPK in human PBMCs revealed an increase in Thr172-phosphorylation of AMPK, confirming target engagement in these blood cells. RNA sequencing of human blood samples identified 608 deregulated genes after AMPK activation. Additionally, AMPK activation led to deregulation of 367 genes in whole blood from HanWistar rats which mapped to the respective human genes. 22 genes out of the intersection of genes deregulated in both species are proposed as potential translatable target engagement biomarker candidates. The most prominent genes were transmembrane glycoprotein NMB (GPNMB, osteoactivin), calcium-binding protein A9 (S100A9), peptidoglycan recognition protein (PGLYRP1) and Ras homolog gene family, member B (RHOB). Specificity for AMPK was shown by testing inactive compound 3 in HanWistar rats. The exposure-effect relationship for GPNMB was investigated in a subchronic study in diabetic ZDF rats. GPNMB showed a dose-dependent up-regulation both acutely and after subchronic dosing. GPNMB up-regulation correlated with an increased Thr172-phosphorylation of AMPK in liver and quadriceps muscle in rats. CONCLUSION: GPNMB has been identified as a translatable target engagement biomarker for use in clinical studies.

Arterial blood pressure and renal sodium excretion in dopamine D3 receptor knockout mice.

Alterations in the dopaminergic system may contribute to the development of hypertension. Recently, it has been reported that pentobarbital-anesthetized mice with deficient dopamine D(3) receptors showed renin-dependent elevation in blood pressure. In a series of experiments, we evaluated the contribution of the dopamine D(3) receptor to the renal sodium excretion and arterial blood pressure behavior in conscious as well as anesthetized dopamine D(3) receptor knockout (-/-) mice. The blood pressure measuring study was designed as a cross-over trial to investigate the influence of different sodium loads. The animals were fed a normal salt diet (0.6% NaCl, NS) for 1 week and afterwards a low (0.2% NaCl, LS) or a high salt diet (4.6% NaCl, HS) for 2 weeks. After the third week, the animals were switched to the corresponding protocol. Systolic blood pressure in conscious (-/-) mice measured by tail-cuff plethysmography was not different from that of wild-type (+/+) animals, irrespective of the time course or the salt diet. In another experiment, challenge of an acute sodium loading per gavage in conscious D(3) receptor (-/-) and (+/+) animals on HS or NS diet did not show significant differences in renal sodium excretion between the two genotypes. Additionally, animals were fed an NS diet for 1 week and an HS diet for another week. As expected, sodium excretion significantly increased after the change from the NS to the HS diet. A slightly lower urinary sodium excretion was observed when comparing D(3) receptor (-/-) mice to their corresponding (+/+) mice, both on an HS diet. Clearance experiments with anesthetized D(3) receptor (-/-) and (+/+) mice were performed to investigate the renal sodium excretion capacity, when exposed to a moderate volume expansion (VE). Urinary sodium excretion increased in response to the VE; however, no difference were observed between the two genotypes. Taking these results together, we conclude that in the present animal model renal dopamine D(3) receptors are not significantly involved in the regulation of blood pressure associated with a deficiency in renal sodium elimination.

A high-throughput, image-based screen to identify kinases involved in brown adipocyte development.

Brown adipose tissue (BAT) is responsible for thermogenesis that is not associated with shivering through the process of converting chemical energy into heat through uncoupling protein 1 (UCP1) in the mitochondria. Thus, expanding or activating BAT could be a potential tool against obesity. To analyze the effect of kinase signaling on brown adipocyte formation, a process that describes the acquisition of the ability to dissipate energy as heat, we performed lentiviral-mediated short hairpin knockdown or used pharmacological inhibitors in a high-content and high-throughput in vitro image-based screen. We identified 190 kinases that either stimulated or inhibited brown adipocyte proliferation, differentiation, or formation. Among these kinases, we found that 5' AMP-activated protein kinase (AMPK) promoted the formation of brown adipocytes abundant inUCP1. Together, our results provide insight into the kinases, particularly AMPK, that regulate brown adipocyte formation.

Absence of amino acid-induced glomerular hyperfiltration in dopamine D3 receptor knockout mice.

Pharmacological inhibition of receptors of the dopamine D2-like family has been shown to abolish the glomerular hyperfiltration in response to amino acids. To further discriminate between the receptor subtypes within the D2-like family, we investigated the effects of amino acid infusion on renal function in dopamine D3 receptor knockout (-/-) mice. In clearance experiments pentobarbital-anesthetized D3 receptor (-/-) and wild-type (+/+) mice were infused with Ringer solution at baseline, followed by a continuous infusion of mixed amino acids (10%). Baseline glomerular filtration rate (GFR), assessed by renal clearance of [3H]-inulin, was the same in D3 receptor (-/-) mice (0.56+/-0.08 ml/min per g kidney weight) and wild-type animals (0.56+/-0.04 ml/min per g kw). During infusion of amino acids, GFR was significantly elevated by 50% in D3 receptor (+/+) mice. In contrast, this amino acid-induced response of GFR was abolished in D3 receptor (-/-) mice. Baseline urinary water and sodium excretion was not significantly different in both groups of mice. As observed in GFR, these renal excretory parameters were significantly elevated during amino acid infusion in D3 receptor (+/+) but not in D3 receptor (-/-) mice. Time controls, constantly infused with Ringer solution, did not show significant changes in GFR, renal water or sodium excretion during the entire experiment, indicating stable experimental conditions. Taken together, the data underline the involvement of dopamine D2-like receptors in the renal response to amino acid infusion and, in addition, attribute this effect to the dopamine D3 receptor subtype.

Short- and Longterm Glycemic Control of Streptozotocin-Induced Diabetic Rats Using Different Insulin Preparations.

The chemical induction of diabetes with STZ has gained popularity because of the relative ease of rendering normal animals diabetic. Insulin substitution is required in STZ-rats in long-term studies to avoid ketoacidosis and consequently loss of animals. Aim of the present studies was to test different insulin preparations and different ways of administration in their ability to reduce blood glucose in STZ-induced diabetic rats. Single dosing of the long-acting insulin analogue glargine was able to dose-dependently reduce blood glucose over 4 h towards normoglycemia in STZ-treated rats. However, this effect was not sustained until 8 h post injection. A more sustained glucose-lowering effect was achieved using insulin-releasing implants. In STZ-rats, 1 insulin implant moderately lowered blood glucose levels 10 days after implantation, while 2 implants induced normoglycemia over the whole day. According to the glucose-lowering effect 1 as well as 2 insulin implants significantly reduced HbA1c measured after 26 days of implantation. In line with the improved glucose homeostasis due to the implants, urinary glucose excretion was also blunted in STZ-treated rats with 2 implants. Since diabetic nephropathy is one of the complications of longterm diabetes, renal function was characterized in the STZ-rat model. Increases in creatinine clearance and urinary albumin excretion resemble early signs of diabetic nephropathy. These functional abnormalities of the kidney could clearly be corrected with insulin-releasing implants 27 days after implantation. The data show that diabetic STZ-rats respond to exogenous insulin with regard to glucose levels as well as kidney parameters and a suitable dose of insulin implants for glucose control was established. This animal model together with the insulin dosing regimen is suitable to address diabetes-induced early diabetic nephropathy and also to study combination therapies with insulin for the treatment of type 1 diabetes.

Effect of dopamine D3 receptor blockade on renal function and glomerular size in diabetic rats.

Dopamine D2-like receptors, including D2, D3, and D4 receptors, are involved in the regulation of glomerular hyperfiltration due to diabetes mellitus. These hemodynamic alterations represent a risk factor for the later development of diabetic nephropathy. The aim of the present study was to determine whether the D3 receptor subtype modulates the diabetes-induced increase in glomerular filtration rate (GFR) in rats. Renal function was studied in Sprague-Dawley rats 14 days after induction of a moderate diabetes mellitus (DM) by streptozotocin and in non-diabetic controls (CON). Rats were orally treated either with the peripherally acting, selective dopamine D3 receptor antagonist BSF 135170 (BSF, 10 mg/kg per day for 2 weeks) or with vehicle (VHC). Perfusion-fixed kidneys were used for estimation of glomerular volume. In conscious rats, which were treated with BSF, the DM-induced increase in fluid intake, urinary output, and renal sodium excretion was significantly less pronounced than in the vehicle group (DM-VHC). In the clearance experiments, GFR in CON was about 0.84+/-0.04 ml/min per 100 g body weight. The DM-VHC group presented a significant glomerular hyperfiltration (1.09+/-0.04 ml/min per 100 g body weight). Treatment with BSF significantly lowered GFR towards levels of CON. The estimated glomerular volume was 0.73+/-0.03 x 10(6) microm3 in the CON-VHC group and 0.86+/-0.04 x 10(6) microm3 in the DM-VHC animals. Interestingly, treatment with BSF decreased the glomerular volume in both groups. Irrespective of BSF treatment, kidney wet weight related to body weight was about 36% higher in DM animals compared with CON animals. We conclude that dopamine D3 receptors represent a target for the modulation of diabetes-induced glomerular hyperfiltration. Therefore, the results encourage the testing of the possible beneficial effects of long-term D3 receptor blockade on the development of diabetic nephropathy.

Subapical localization of the dopamine D3 receptor in proximal tubules of the rat kidney.

The dopamine D3 receptor (D3R), intensively studied in neuroscience, also plays an important role in the regulation of renal and cardiovascular function. In contrast to functional findings, less information is available on its localization in the kidney. Neither RT-PCR studies nor radioligand binding assays are suitable to selectively determine the distribution of renal D3R at the level of cellular or even subcellular structures. We studied the renal D3R distribution in Sprague-Dawley rats by a polyclonal antiserum directed against an epitope in the third intracytoplasmic loop. D3R immunoreactivity was detected by indirect immunofluorescence and confocal laser scanning microscopy. D3R staining was confined to the renal cortex and occurred in proximal convoluted tubules near or in direct connection with the urinary pole of the glomeruli. The fluorescent spots were restricted to the subapical portion of the proximal tubular cells. Double staining with the F-actin marker phalloidin revealed a localization of the D3R below the brush border region. However, staining by anti-beta1/beta2-adaptins, recognizing clathrin-coated compartments, did not correspond to the distribution of the D3R signal. This is the first description of a D3R accumulation in a cytoplasmic pool in the kidney, probably corresponding to a recycling mechanism or storage compartment.

Tissue levels of S-adenosylhomocysteine in the rat kidney: effects of ischemia and homocysteine.

Most S-adenosylmethionine (AdoMet)-dependent methyltransferases are regulated in vivo by the AdoMet/S-adenosylhomocysteine (AdoHcy) ratio, also termed as "methylation potential." Since adenosine inhibits in vitro AdoHcy hydrolysis and since adenosine tissue levels increase during hypoxia, it can be predicted that AdoHcy levels may increase in the rat kidney in parallel of those of adenosine. Therefore, the present investigation was performed to assess changes of renal AdoHcy and AdoMet tissue contents during ischemia and after administration of adenosine and homocysteine or both in the ischemic rat kidney. In anesthetized rats ischemia of the kidney was induced by renal artery occlusion for various time intervals. Adenosine and homocysteine were infused into the renal artery of the ischemic kidney. To induce a hyperhomocysteinemia homocysteine was continuously infused. The kidneys were removed and immediately snap-frozen. Tissue contents of AdoHcy, AdoMet, adenosine and adenine nucleotides were analyzed by means of HPLC. Under normoxic condition the tissue contents of AdoHcy, AdoMet and adenosine were 0.7+/-0.05, 44.1+/-1.0 and 3.8+/-0.1nmol/g wet weight, respectively. Renal ischemia for 30min resulted in an increase of AdoHcy levels from 0.7+/-0.05 to 9.1+/-0.6nmol/g wet weight and in a dramatic decrease of the AdoMet/AdoHcy ratio and energy charge from 65.1+/-5.6 to 2.8+/-0.2 and from 0.87+/-0.01 to 0.25+/-0.01, respectively. Application of exogenous adenosine into the ischemic kidney did not result in further AdoHcy accumulation. However, when homocysteine was infused into the ischemic kidney, AdoHcy increased five-fold above control levels, during 5min ischemia. Systemic infusion of homocysteine leads to a reduction of the methylation potential also in the normoxic kidney. We conclude that (i) the methylation potential in the kidney is markedly reduced during ischemia, mainly due to accumulation of AdoHcy; (ii) elevation of AdoHcy tissue content during ischemia is the result of the inhibition of AdoHcy hydrolysis; (iii) homocysteine is rate limiting for AdoHcy synthesis in the ischemic kidney; (iv) under normoxic conditions hyperhomocysteinemia can affect the methylation potential in the renal tissue.

Dopamine D3 receptor mRNA and renal response to D3 receptor activation in spontaneously hypertensive rats.

Defective dopamine receptors may be involved in the development of hypertension. Recently, it has been shown that gene expression and function of the renal dopamine D3 receptor is impaired in salt-sensitive Dahl rats, a model of salt-dependent hypertension. Here, the functional response to D3 receptor activation was investigated in spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto rats (WKY). In addition, expression of the D3 receptor gene was studied in both rat strains. In clearance experiments, Ringer solution was infused at baseline in thiopental-anesthetized SHR and WKY (each n = 8), followed by an infusion of R(+)-7-hydroxy-dipropylaminotetralin (DPAT), a specific D3 receptor agonist. DPAT was infused in two consecutive doses of 0.01 and 0.1 microg/min per kg body weight. During the entire experiment mean arterial blood pressure was significantly higher (1.5-fold) in adult SHR when compared to age-matched WKY. In both groups DPAT infusion induced a similar dose-dependent increase in urinary flow rate and sodium excretion by a maximum of 2.3-fold and 3.5-fold, respectively. DPAT also increased the glomerular filtration rate in both SHR and WKY. Reverse transcription-polymerase chain reaction studies of whole kidney samples showed no significant differences between young prehypertensive and adult hypertensive SHR when compared to age-matched normotensive WKY. In summary, pharmacological dopamine D3 receptor activation induces a uniform renal response in SHR and WKY. Together with the similar D3 receptor gene expression in both rat strains, which is independent of age or blood pressure levels, the results do not support the notion that the dopamine D3 receptor system is involved in the pathogenesis of hypertension in the SHR model.

Role of the renin-angiotensin system in the compensation of quinpirole-induced blood pressure decrease.

The dopamine D(2)-like receptor agonist quinpirole has been reported to lower blood pressure. This effect appears to be mediated via activation of presynaptic D(2)-like receptors inhibiting the stimulated neural norepinephrine release. The aim of the present study was to investigate the role of renal nerves and the renin-angiotensin system (RAS) in the blood pressure lowering effect of quinpirole. Therefore, clearance experiments using different doses of quinpirole (0.3 to 100 microg/kg/min) were performed in thiopental-anesthetized rats with intact kidneys (INN) or 5 to 7 days after bilateral renal denervation (DNX). The functional involvement of the RAS in the blood pressure lowering effect of quinpirole was determined in rats pretreated with a subpressor dose of angiotensin II (10 microg/kg/min) or in rats pretreated with the angiotensin II (AT(1)) receptor antagonist losartan, in a subdepressor dose (10 microg/kg/min). Quinpirole dose-dependently decreased mean arterial blood pressure (MAP) by up to 29%. This blood pressure lowering effect of quinpirole was observed at lower doses in DNX rats when compared with INN animals (ED(50): 0.98 microg/kg/min in DNX vs. 6.02 microg/kg/min in INN animals). Quinpirole in a dose of 3 microg/kg/min, which did not affect MAP in vehicle treated INN rats, significantly reduced MAP in rats with losartan pretreatment. In DNX rats pretreated with angiotensin II the MAP-response to the infusion of 3 microg/kg/min quinpirole was clearly attenuated in comparison with untreated DNX animals. Our data show that stimulation of dopamine D(2)-like receptors dose-dependently decreased blood pressure, which was potentiated by both interruption of the renal innervation and AT(1) receptor blockade, while exogenous ANG II restored the enhancement of the blood pressure response to quinpirole. We conclude that the increased vasodilatory effect of quinpirole after renal denervation might depend on a decreased activity of the RAS.

Characterisation of cerivastatin as a P-glycoprotein substrate: studies in P-glycoprotein-expressing cell monolayers and mdr1a/b knock-out mice.

The aim of this study was to characterise the role of the efflux transporter P-glycoprotein in the disposition of cerivastatin. We investigated directional transport characteristics of [14C]cerivastatin across cell monolayers expressing P-glycoprotein (Caco-2 and L-MDR1) and disposition of cerivastatin in mice with disrupted mdr1a and mdr1b genes. The mice were given orally 1 mg/kg cerivastatin and plasma and tissue samples for analysis of cerivastatin were obtained 10, 20, or 30 min after drug administration. Four knock-out mice and four wild-type mice were studied at each time point. In addition, the hypothesis that gemfibrozil-mediated inhibition of P-glycoprotein contributes to the interaction between gemfibrozil and cerivastatin was tested in Caco-2 cells. The apparent permeability coefficient (P(app)) value for the basal-to-apical transport of cerivastatin in Caco-2 and L-MDR1 cell monolayers was 2.4 times (P<0.001) and 3.8 times (P<0.001) as high as the apical-to-basal P(app) value respectively. The P-glycoprotein inhibitor PSC-833 (1 microM) inhibited the net basal-to-apical transport of cerivastatin in Caco-2 monolayers by 35% (P<0.01) and the MRP inhibitor MK-571 (10 microM) by 50% (P<0.01). At concentrations up to 250 microM, gemfibrozil showed no significant effects on the net transport of cerivastatin in Caco-2 cells. The concentration of cerivastatin in the brain at 30 min was 3.1 times higher in the knock-out mice than in the wild-type mice (P<0.05). The brain-to-plasma cerivastatin concentration ratio at 20 min and 30 min was 2.1 (P<0.05) and 3.6 times (P<0.05) higher respectively in the knock-out animals compared with the wild-type animals. Collectively, these results indicate that cerivastatin is a P-glycoprotein substrate, although other transporters probably contribute to cerivastatin transport in humans. As several statins are P-glycoprotein substrates, beneficial as well as adverse effects of the statins might be affected by interindividual differences in P-glycoprotein expression or function caused by, e.g., the MDR1 polymorphism.

An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

Potent cholesteryl ester transfer protein inhibitors of reduced lipophilicity: 1,1'-spiro-substituted hexahydrofuroquinoline derivatives.

A series of 1,1'-spiro-substituted hexahydrofuroquinoline derivatives exhibiting potent cholesteryl ester transfer protein (CETP) inhibition at reduced lipophilicity was identified. A focused structure-activity relationship (SAR) exploration led to the potent and comparatively polar CETP inhibitor 26 showing robust high density lipoprotein-cholesterol (HDL-C) elevation and low density lipoprotein-cholesterol (LDL-C) reduction in transgenic hCETP/hApoB-100 mice. Compound 26 was also shown to positively differentiate from highly lipophilic CETP inhibitors in its complete elimination from fat tissue in hCETP transgenic mice as evident within 21 days after cessation of treatment. In addition, compound 26 showed no significant effects on aldosterone secretion from H295R cells, as well as no significant effects on blood pressure and electrocardiogram parameters in telemetrized cynomolgus monkeys.

Gene delivery to adipose tissue using transcriptionally targeted rAAV8 vectors.

In recent years, the increasing prevalence of obesity and obesity-related co-morbidities fostered intensive research in the field of adipose tissue biology. To further unravel molecular mechanisms of adipose tissue function, genetic tools enabling functional studies in vitro and in vivo are essential. While the use of transgenic animals is well established, attempts using viral and non-viral vectors to genetically modify adipocytes in vivo are rare. Therefore, we here characterized recombinant Adeno-associated virus (rAAV) vectors regarding their potency as gene transfer vehicles for adipose tissue. Our results demonstrate that a single dose of systemically applied rAAV8-CMV-eGFP can give rise to remarkable transgene expression in murine adipose tissues. Upon transcriptional targeting of the rAAV8 vector to adipocytes using a 2.2 kb fragment of the murine adiponectin (mAP2.2) promoter, eGFP expression was significantly decreased in off-target tissues while efficient transduction was maintained in subcutaneous and visceral fat depots. Moreover, rAAV8-mAP2.2-mediated expression of perilipin A - a lipid-droplet-associated protein - resulted in significant changes in metabolic parameters only three weeks post vector administration. Taken together, our findings indicate that rAAV vector technology is applicable as a flexible tool to genetically modify adipocytes for functional proof-of-concept studies and the assessment of putative therapeutic targets in vivo.

Identification of a potential biomarker for FABP4 inhibition: the power of lipidomics in preclinical drug testing.

The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4, which is almost exclusively expressed in adipocytes and macrophages, contributes to the development of insulin resistance and atherosclerosis in mice. Lack of FABP4 protects against the development of insulin resistance associated with genetic or diet-induced obesity in mice. Furthermore, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in apolipoprotein E-deficient mice. The FABP4 small-molecule inhibitor BMS309403 has demonstrated efficacy in mouse models for type 2 diabetes mellitus and atherosclerosis, resembling phenotypes of mice with FABP4 deficiency. However, despite the therapeutically attractive long-term effects of FABP4 inhibition, an acute biomarker for drug action is lacking. The authors applied mass spectrometry lipidomics analysis to in vitro and in vivo (plasma and adipose tissue) samples upon inhibitor treatment. They report the identification of a potential biomarker for acute in vivo FABP4 inhibition that is applicable for further investigations and can be implemented in simple and fast-flow injection mass spectrometry assays. In addition, this approach can be considered a proof-of-principle study that can be applied to other lipid-pathway targeting mechanisms.

Evidence for a predominant intrinsic sympathetic control of cerebral blood flow alterations in an animal model of cerebral arteriovenous malformation.

In terms of neurogenic cerebral blood flow (CBF) control, the activity of the sympathetic nervous system (SNS) has a regulating effect. The impact of a manipulation of both the peripheral (via the perivascular sympathetic net) and central components (via the intracortical noradrenergic terminals originating from the locus coeruleus) on CBF-and especially on hyperperfusion syndromes-is unclear. To test the specific patterns following such alterations, cortical oxygen saturation (rSO2), regional CBF (rCBF), and cortical interstitial norepinephrine (NE) concentrations were measured. Twelve weeks after either the creation of an extracranial AV fistula or sham operation, 80 male Sprague-Dawley rats underwent one of the following procedures: (1) no SNS manipulation, (2) peripheral SNS inhibition via bilateral sympathectomy, (3) central SNS inhibition via the neurotoxin DSP-4, or (4) complete SNS inhibition. Norepinephrine concentrations were lowest after complete inhibition (NE [nmol]: pre, 1.8 ± 1.2; post, 2.4 ± 1.8) and highest following peripheral inhibition (NE [nmol]: pre, 3.6 ± 1.9; post, 6.6 ± 4.4). Following fistula occlusion, rCBF (laser Doppler unit [LDU]) and rSO2 (%SO2) increases were highest after complete inhibition (pre: 204 ± 14 LDU, 34 ± 3%SO2; post: 228 ± 18 LDU, 39 ± 3%SO2) and lowest after peripheral inhibition (pre: 221 ± 18 LDU, 41 ± 2%SO2; post: 226 ± 14 LDU, 47 ± 2%SO2). Thus, a complete inhibition down-regulates SNS activity and provokes a cortical hyperperfusion condition. With this, the hitherto unknown predominant role of the intrinsic component could be demonstrated for the first time in vivo.