Search

Doxorubicin is a potent anticancer drug with cardiotoxicity hampering its use. Neuropeptide Y (NPY) is the most abundant neuropeptide in the heart and a co-transmitter of the sympathetic nervous system that plays a role in cardiac diseases. The aim of this work was to study the impact of NPY on doxorubicin-induced cardiotoxicity. Transgenic mice overexpressing NPY in noradrenergic neurons (NPY-OEDβH) and wild-type mice were treated with a single dose of doxorubicin. Doxorubicin caused cardiotoxicity in both genotypes as demonstrated by decreased weight gain, tendency to reduced ejection fraction, and changes in the expression of several genes relevant to cardiac pathology. Doxorubicin resulted in a tendency to lower ejection fraction in NPY-OEDβH mice more than in wild-type mice. In addition, gain in the whole body lean mass gain was decreased only in NPY-OEDβH mice, suggesting a more severe impact of doxorubicin in this genotype. The effects of doxorubicin on genes expressed in the heart were similar between NPY-OEDβH and wild-type mice. The results demonstrate that doxorubicin at a relatively low dose caused significant cardiotoxicity. There were differences between NPY-OEDβH and wild-type mice in their responses to doxorubicin that suggest NPY to increase susceptibility to cardiotoxicity. This may point to the therapeutic implications as suggested for NPY system in other cardiovascular diseases. Electronic supplementary material The online version of this article (10.1007/s12012-019-09557-2) contains supplementary material, which is available to authorized users.

Adipose tissue macrophages (ATMs) regulate homeostasis and contribute to the metabolically harmful chronic inflammation in obese individuals. While evident heterogeneity of resident ATMs has been described previously, their phenotype, developmental origin, and functionality remain inconsistent. We analyzed white adipose tissue (WAT) during homeostasis and diet interventions using comprehensive and unbiased single-cell mass cytometry and genetic lineage tracking models. We now provide a uniform definition of individual subsets of resident ATMs. We show that in lean mice, WAT co-harbors eight kinetically evolving CD206+ macrophage subpopulations (defined by TIM4, CD163, and MHC II) and two CD206– macrophage subpopulations. TIM4–CD163+, TIM4–CD163– and CD206– macrophage populations are largely bone marrow-derived, while the proliferating TIM4+CD163+ subpopulation is of embryonic origin. All macrophage subtypes are active in phagocytosis, endocytosis, and antigen processing in vitro, whereas TIM4+CD163+ cells are superior in scavenging in vivo. A high-fat diet induces massive infiltration of CD206– macrophages and selective down-regulation of MHC II on TIM4+ macrophages. These changes are reversed by dietary intervention. Thus, the developmental origin and environment jointly regulate the functional malleability of resident ATMs.

γ-Melanocyte stimulating hormone (γ-MSH) is an endogenous agonist of the melanocortin 3-receptor (MC3R). Genetic disruption of MC3Rs increases adiposity and blunts responses to fasting, suggesting that increased MC3R signaling could be physiologically beneficial in the long term. Interestingly, several studies have concluded that activation of MC3Rs is orexigenic in the short term. Therefore, we aimed to examine the short- and long-term effects of γ-MSH in the hypothalamic arcuate nucleus (ARC) on energy homeostasis and hypothesized that the effect of MC3R agonism is dependent on the state of energy balance and nutrition. Lentiviral gene delivery was used to induce a continuous expression of γ-Msh only in the ARC of male C57Bl/6N mice. Parameters of body energy homeostasis were monitored as food was changed from chow (6 weeks) to Western diet (13 weeks) and back to chow (7 weeks). The γ-MSH treatment decreased the fat mass to lean mass ratio on chow, but the effect was attenuated on Western diet. After the switch back to chow, an enhanced loss in weight (−15% vs −6%) and fat mass (−37% vs −12%) and reduced cumulative food intake were observed in γ-MSH-treated animals. Fasting-induced feeding was increased on chow diet only; however, voluntary running wheel activity on Western diet was increased. The γ-MSH treatment also modulated the expression of key neuropeptides in the ARC favoring weight loss. We have shown that a chronic treatment intended to target ARC MC3Rs modulates energy balance in nutritional state-dependent manner. Enhancement of diet-induced weight loss could be beneficial in treatment of obesity.

Supplemental Digital Content is available in the text., Objective— The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates anti-inflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis. Approach and Results— Apoe−/− (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1re/e) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe−/− Mc1re/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe−/− controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe−/− Mc1re/e mice showed a defect in bile acid metabolism that aggravated high-fat diet–induced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6Chigh monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6Chigh monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1. Conclusions— The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation.

The commonly used sedative alpha(2)-adrenoceptor agonist dexmedetomidine has adverse cardiovascular effects in dogs that can be prevented by concomitant administration of the peripherally acting alpha(2)-adrenoceptor antagonist MK-467. An ancillary effect of dexmedetomidine is to decrease insulin release from the pancreas, whereas MK-467 stimulates insulin release. This study assessed the effects of co-administered dexmedetomidine and MK-467 in a canine glibenclamide-induced hypoglycaemia model. In a randomised, cross-over experiment, eight beagle dogs received five intravenous treatments, comprising two administrations of saline, with dexmedetomidine or dexmedetomidine and MK-467, and three administrations of glibenclamide, with saline, dexmedetomidine or dexmedetomidine and MK-467. Plasma concentrations of glucose, lactate, insulin, glucagon and the test drugs were monitored. Administration of glibenclamide significantly increased insulin secretion and decreased blood glucose concentrations. Dexmedetomidine counteracted glibenclamide-evoked hypoglycaemia. This was opposed by the alpha(2)-adrenoceptor antagonist MK-467, but the glibenclamide-evoked hypoglycaemia was not potentiated by co-administration of dexmedetomidine and MK-467. None of the dogs developed uncontrolled hypoglycaemia. Thus, the combination of dexmedetomidine and MK-467 appeared to be safe in this canine hypoglycaemia model. Nevertheless, when MK-467 is used to alleviate the undesired cardiovascular effects of alpha(2)-adrenoceptor agonists in dogs, it should be used with caution in animals at risk for hypoglycaemia because of its insulin-releasing and hypoglycaemic effects. (C) 2018 The Author(s). Published by Elsevier Ltd.

Atherosclerosis is a chronic inflammatory disease of the arteries. The disease is initiated by endothelial dysfunction that allows the transport of leukocytes and low-density lipoprotein into the vessel wall forming atherosclerotic plaques. The melanocortin system is an endogenous peptide system that regulates, for example, energy homeostasis and cardiovascular function. Melanocortin treatment with endogenous or synthetic melanocortin peptides reduces body weight, protects the endothelium and alleviates vascular inflammation, but the long-term effects of melanocortin system activation on atheroprogression remain largely unknown. In this study, we evaluated the effects of transgenic melanocortin overexpression in a mouse model of atherosclerosis. Low-density lipoprotein receptor-deficient mice overexpressing alpha- and gamma3-MSH (MSH-OE) and their wild-type littermates were fed either a regular chow or Western-style diet for 16 weeks. During this time, their metabolic parameters were monitored. The aortae were collected for functional analysis, and the plaques in the aortic root and arch were characterised by histological and immunohistochemical stainings. The aortic expression of inflammatory mediators was determined by quantitative PCR. We found that transgenic MSH-OE improved glucose tolerance and limited atherosclerotic plaque formation particularly in Western diet-fed mice. In terms of aortic vasoreactivity, MSH-OE blunted alpha1-adrenoceptor-mediated vasoconstriction and enhanced relaxation response to acetylcholine, indicating improved endothelial function. In addition, MSH-OE markedly attenuated Western diet-induced upregulation of proinflammatory cytokines (Ccl2, Ccl5 and Il6) that contribute to the pathogenesis of atherosclerosis. These results show that the activation of the melanocortin system improves glucose homeostasis and limits diet-induced vascular inflammation and atherosclerotic plaque formation.

Objective— Melanocortin peptides have been shown to elicit anti-inflammatory actions and to promote vascular endothelial function by activating type 1 and 3 melanocortin receptors. Here, we addressed whether these favorable properties of melanocortins could reduce atherosclerotic plaque inflammation and improve vasoreactivity in atherosclerotic mice. Approach and Results— Low-density lipoprotein receptor–deficient mice expressing only apolipoprotein B100 were fed a high-fat diet for 8 or 16 weeks and treated with either vehicle or a stable melanocortin analog, melanotan II (MT-II, 0.3 mg/kg per day, 4 weeks). We determined plaque uptake of fluorine-18–labeled fluorodeoxyglucose as a surrogate marker for atherosclerotic plaque inflammation and vascular function of the aorta by ex vivo analyses. MT-II had no effect on body weight or composition, or plasma cholesterol levels in atherosclerotic mice. Without attenuating atherosclerotic lesion size or lesional macrophage accumulation, MT-II treatment reduced fluorine-18–labeled fluorodeoxyglucose uptake in the atherosclerotic plaques. Resident macrophages in the lesions of MT-II–treated mice were polarized toward the anti-inflammatory M2 phenotype. Systemic inflammation was also attenuated by MT-II intervention as evidenced by decreased plasma levels of proinflammatory cytokines. In terms of aortic vasoreactivity, MT-II–treated mice showed enhanced endothelium-dependent relaxations, as well as promotion of vascular sensitivity to nitric oxide–mediated vasodilation, which were markedly impaired in control mice after prolonged duration of diet exposure. Conclusions— The present study demonstrates that pharmacological activation of the melanocortin system has therapeutic benefits in pre-established atherosclerosis by limiting plaque inflammation and promoting vascular endothelial function, which may provide a novel therapeutic approach for atherosclerosis.

Melanocyte stimulating hormone (MSH) derived from the pro-hormone pro-opiomelanocortin (POMC) has potent effects on metabolism and feeding that lead to reduced body weight in the long-term. To determine the individual roles of POMC derived peptides and their sites of action, we created a method for the delivery of single MSH peptides using lentiviral vectors and studied the long-term anti-obesity effects of hypothalamic α-MSH overexpression in mice. An α-MSH lentivirus (LVi-α-MSH-EGFP) vector carrying the N'-terminal part of POMC and the α-MSH sequence was generated and shown to produce bioactive peptide in an in vitro melanin synthesis assay. Stereotaxis was used to deliver the LVi-α-MSH-EGFP or control LVi-EGFP vector to the arcuate nucleus (ARC) of the hypothalamus of male C57Bl/6N mice fed on a high-fat diet. The effects of 6-week-treatment on body weight, food intake, glucose tolerance and organ weights were determined. Additionally, a 14-day pairfeeding study was conducted to assess whether the weight decreasing effect of the LVi-α-MSH-EGFP treatment is dependent on decreased food intake. The 6-week LVi-α-MSH-EGFP treatment reduced weight gain (8.4 ± 0.4 g versus 12.3 ± 0.6 g; P < 0.05), which was statistically significant starting from 1 week after the injections. The weight of mesenteric fat was decreased and glucose tolerance was improved compared to LVi-EGFP treated mice. Food intake was decreased during the first week in the LVi-α-MSH-EGFP treated mice but subsequently increased to the level of LVi-EGFP treated mice. The LVi-EGFP injected control mice gained more weight even when pairfed to the level of food intake by LVi-α-MSH-EGFP treated mice. We demonstrate that gene transfer of α-MSH, a single peptide product of POMC, into the ARC of the hypothalamus, reduces obesity and improves glucose tolerance, and that factors other than decreased food intake also influence the weight decreasing effects of α-MSH overexpression in the ARC. Furthermore, viral MSH vectors delivered stereotaxically provide a novel tool for further exploration of chronic site-specific effects of POMC peptides.

To study the effects of rapid i.v. glucose bolus on insulin, leptin, ghrelin, peptide YY (PYY), free fatty acids (FFA), glucagon and glucagon-like peptide-1 (GLP-1) concentrations together with self-reported satiety ratings in lean and obese human subjects.Twenty-five healthy subjects were recruited, 12 were lean (mean age = 26 years, BMI range = 19.8-23.9 kg/m(2)) and 13 were obese (mean age = 27 years, BMI range = 27.7-42.2 kg/m(2)). In two separate 55 min counter-balanced blinded sessions (separate days), subjects were administered an i.v. dose of 300 mg/kg glucose or saline. Blood concentrations of several feeding-related hormones were recorded at multiple time points, together with ratings of satiety and euphoria.Greater increases in glucose concentrations were observed in the obese group compared to the lean group (p0.0001). In both lean and obese subjects, glucose injection induced a clear fall in the concentrations of FFA, ghrelin, glucagon and PYY (p0.0001) but not in the concentrations of leptin or GLP-1. Obese subjects showed positive correlations between satiety and glucose, but only at time points 30 min (r = 0.73, p = 0.005) and 55 min (r = 0.82, p = 0.0005).The directions and the magnitudes of short-term hormonal changes after i.v. glucose challenge are the same in lean and moderately obese subjects. Possible short-term regulatory effects of leptin and GLP-1 can not be induced by acute energy load bypassing the GI-tract.

Alpha(2)-Adrenoceptors regulate insulin secretion and sympathetic output. In the present study, alpha(2A)-adrenoceptor knockout (alpha(2A)-KO) mice and their C57BL/6J wild-type (WT) controls were used to assess the glucoregulatory role of the alpha(2A)-adrenoceptor subtype in vivo. Fasting and glucose-stimulated blood glucose and plasma insulin levels were determined with or without (+/-)-propranolol (5 mg/kg) or atropine (10 mg/kg) pre-treatment. Intraperitoneal glucose (1 g/kg) and insulin (0.5 and 1.0 IU/kg) tolerance tests were performed. Fasting plasma glucagon and corticosterone levels were measured. Blood glucose levels (mean+/-S.E.M.) were lower in alpha(2A)-KO males (7.2+/-0.6 mM) and females (7.2+/-0.2 mM) than in WT males (9.8+/-0.3 mM) and females (9.1+/-0.3 mM). Plasma insulin levels were higher in alpha(2A)-KO males (2.2+/-0.5 microg/l) and females (1.7+/-0.3 microg/l) than in WT males (0.7+/-0.1 microg/l) and females (0.8+/-0.2 microg/l). These differences remained after pharmacological beta-adrenoceptor and muscarinic acetylcholine receptor inhibition. In spite of a tendency for slightly decreased insulin sensitivity in alpha(2A)-KO mice, glucose tolerance in alpha(2A)-KO mice was significantly better than in WT mice. However, glucose-stimulated insulin secretion was not increased in alpha(2A)-KO mice compared to WT controls. Plasma glucagon levels, but not corticosterone levels, were elevated in alpha(2A)-KO mice. These results suggest that lack of inhibitory pancreatic beta-cell alpha(2A)-adrenoceptor function results in hyperinsulinaemia, reduced blood glucose levels and improved glucose tolerance in alpha(2A)-KO mice, and demonstrate a key role for the alpha(2A)-adrenoceptor in adrenergic regulation of blood glucose and insulin homeostasis.

Dopamine is known to regulate food intake by modulating food reward via the mesolimbic circuitry of the brain. The objective of this study was to compare the effects of high energy input (i.v. glucose) on striatal and thalamic dopamine release in overweight and lean individuals. We hypothesized that glucose would induce dopamine release and positive ratings (e.g., satiety) in Behavioral Analog Scales, particularly in food-deprived lean subjects. [C-11]raclopride PET was performed for 12 lean (mean BMI = 22 kg/m(2)) and 12 overweight (mean BMI = 33 kg/m(2)) healthy subjects. Each subject was imaged twice in a blinded counter-balanced setting, after 300 mg/kg i.v. glucose and after i.v. placebo. Dopamine D2 receptor binding potentials (BPs) were estimated. The voxel-based analysis of the baseline scans indicated lower striatal. BPs in the overweight group and a negative correlation between BMIs and BPs. Intravenous glucose did not have a significant effect on BPs in overweight or lean subjects (male and female groups combined). However, BP changes were opposite in the two gender groups. In male subjects, significant BP reductions after glucose were seen in the right and left caudate nucleus, left putamen, and right thalamus. In female subjects, increases in BP secondary to glucose were seen in the right caudate nucleus and right and left putamen. The sexually dimorphic effect of glucose was seen in both overweight and lean subjects. Although gender differences were not among the a priori hypotheses of the present study and, therefore, they must be considered to be preliminary findings, we postulate that this observation is a reflection of an interaction between glucose, sex steroids (estrogen), leptin, and dopamine.

Laboratory rats are generally fed ad libitum, although this method is associated with obesity and an increased frequency of spontaneous tumours. It has been challenging looking for ways to limit feed consumption in group-housed rats without any setbacks to animal welfare and scientific results. The diet board, as a method of dietary restriction, was used in the present study. Diet board feeding allows group housing and should result in enhanced welfare compared with traditional methods of dietary restriction. With respect to animal model robustness and translatability of results it is important that the feeding regime does not affect diurnal rhythmicity of biological parameters. In the present study the effects of diet board feeding on diurnal rhythms of blood glucose, serum ghrelin, faecal immunoglobulin A (IgA) and faecal corticosterone were assessed. The diet board did not alter diurnal rhythms, and adds weight to the use of this method for dietary restriction which should benefit animal health and the validity of scientific results generated from the animals.

Glucocorticoids regulate body energy balance through both peripheral and central mechanisms. In order to understand the central mechanisms that mediate these effects of glucocorticoids we studied the effects of adrenalectomy (ADX) and food deprivation on the expression of four neuropeptide genes (measured by S1 nuclease protection assay) in the medial basal hypothalamus (MBH), which are known to regulate energy balance: pro-opiomelanocortin (POMC), agouti-related peptide (AGRP), neuropeptide Y (NPY), and cocaine and amphetamine regulated transcript (CART). Adult male rats were ADX or sham operated (SHAM), and studied 1-2 weeks later. In the first study effects of ADX and corticosterone replacement on POMC and AGRP expression were determined. ADX decreased POMC and AGRP gene expression in the MBH by 27 and 38%, respectively, compared to SHAM rats. Corticosterone treatment increased the expression of POMC by 87% and AGRP by 45% in ADX rats. The second study was designed to determine if glucocorticoids are necessary for the fasting induced changes in POMC, AGRP, NPY and CART in the MBH. ADX caused a 20-30% decrease in the expression of all four neuropeptide genes in the MBH. As expected, fasting suppressed POMC and CART expression and increased AGRP and NPY expression. The fasting-induced increases in AGRP and NPY persisted after ADX but no further significant decreases in POMC or CART were noted after fasting in ADX rats. Plasma leptin and insulin declined significantly after ADX and increased with corticosterone replacement; both leptin and insulin declined further in fasted, ADX animals. In conclusion, ADX decreases both anorexigenic, POMC and CART, and orexigenic, AGRP and NPY, neuropeptide gene expression in the MBH. AGRP and NPY decrease after ADX despite the fall in plasma leptin and insulin concentrations which in other situations would increase these neuropeptides. Furthermore, glucocorticoids are not required for fasting-induced upregulation of AGRP and NPY expression.

The POMC pathway is involved in the regulation of energy and cardiovascular homeostasis in the hypothalamus and the brain stem. Although the acute effects of POMC-derived peptides in different brain locations have been elucidated, the chronic site-specific effects of distinct peptides remain to be studied. To this end, we used a lentiviral gene delivery vector to study the long-term effects of α-MSH in the nucleus tractus solitarius (NTS) of the brain stem. The α-MSH vector (LVi–α-MSH–EGFP) based on the N-terminal POMC sequence and a control vector (LVi–EGFP) were delivered into the NTS of C57BL/6N male mice fed on a western diet. Effects on body weight and composition, feeding, glucose metabolism, and hemodynamics by telemetric analyses were studied during the 12-week follow-up. The LVi–α-MSH–EGFP-treated mice had a significantly smaller gain in the fat mass compared with LVi–EGFP-injected mice. There was a small initial decrease in food intake and no differences in the physical activity. Glucose metabolism was not changed compared with the control. LVi–α-MSH–EGFP increased the heart rate (HR), which was attenuated by adrenergic blockade suggesting an increased sympathetic activity. Reduced response to muscarinic blockade suggested a decreased parasympathetic activity. Fitting with sympathetic activation, LVi–α-MSH–EGFP treatment reduced urine secretion. Thus, the results demonstrate that long-term α-MSH overexpression in the NTS attenuates diet-induced obesity. Modulation of autonomic nervous system tone increased the HR and most probably contributed to an anti-obesity effect. The results underline the key role of NTS in the α-MSH-induced long-term effects on adiposity and in regulation of sympathetic and parasympathetic activities.

Celiprolol is a novel beta-adrenoceptor blocking drug that displays clinically favorable effects on glucose and lipid metabolism. Because some other atypical beta-adrenoceptor blocking drugs have been described to act as agonists on beta(3)-adrenoceptors, we aimed to investigate the effects of celiprolol on brown fat and beta(3)-adrenoceptors. Chronic treatment of obese fa/fa Zucker rats with celiprolol (50 mg/kg/day orally for 20 days) increased GDP binding to brown fat mitochondria by 1.5-fold, whereas beta(3)-adrenoceptor agonist ZD7114 ((S)-4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]-N-(2-methoxyet hyl )phenoxyacetamide, 3 mg/kg/day) increased the binding by 3.3-fold. Weight gain was reduced by 19% due to decreased water and food intakes in celiprolol-treated rats. Celiprolol did not activate lipolysis in rat adipocytes in vitro or stimulate human beta(3)-adrenoceptors expressed in Chinese hamster ovary cells as measured with Cytosensor microphysiometer. Therefore, celiprolol does not seem to activate brown fat via beta(3)-adrenoceptors.

The expressions of uncoupling proteins 2 and 3 (UCP2; UCP3) mRNA were studied in obese (fa/fa) Zucker rats treated with two weight gain reducing agents for three weeks. The specific beta 3-adrenoceptor agonist BRL 35135 (0.5 mg/kg/day orally) increased the expression of UCP3 mRNA by 3.8-fold (P0.0001; two-way ANOVA) and that of UCP1 mRNA by 2.6-fold (P = 0.014) in brown adipose tissue, but had no effect on expression of UCP3 mRNA in white fat or in the soleus muscle, or on UCP2 mRNA expression in brown or white fat. The antihyperglycemic metformin (300 mg/kg/day orally) had no effect on expressions of UCP1, UCP2 or UCP3 in any tissue studied. Concentrations of plasma insulin were significantly correlated with the levels of white fat UCP2 mRNA (in the control group: r = 0.89, P = 0.0015) and UCP3 mRNA (in the control group: r = 0.80, P = 0.009) suggesting that insulin may play a role in the control of UCP2 and UCP3 mRNA expressions in white adipose tissue.

MPV-1743 A III ((+/-)-4-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) is a novel imidazoline derivative. In this study, it was shown to bind with high affinity to alpha2-adrenoceptor subtypes alpha2A (IC50) = 0.66 +/- 0.06 nM), alpha2B (IC50) = 3.8 +/- 0.53 nM), alpha2C (IC50) = 3.1 +/- 0.61 nM) in the recombinant S115 cells and to alpha2D (IC50 = 0.94 +/- 0.10 nM) in the rat submandibular gland. MPV-1743 A III also showed remarkably high affinity to alpha1-adrenoceptors (IC50 = 150 +/- 12 nM) in the rat cerebral cortex and to imidazoline I2b-binding sites (IC50) = 150 +/- 5.0 nM) in the rat liver. The functional alpha2-adrenoceptor antagonistic effect of MPV-1743 A III was demonstrated by studying the ability of orally administered MPV-1743 A III to reverse and prevent the alpha2-adrenoceptor agonist detomidine-induced mydriasis in rat. The anti-obesity effect of MPV-1743 A III was investigated in genetically obese (fa/fa) Zucker rats in two different phases of obesity. Chronic treatment with MPV-1743 A III (0.3 3 mg/kg per day p.o. for 3 weeks) dose dependently decreased weight gain in early-phase obesity. In fully established obesity, GDP binding to mitochondria and expression of uncoupling protein mRNA were increased in brown adipose tissue by MPV-1743 A III indicating an activation of non-shivering thermogenesis. The present study shows that MPV- 1743 A III has a modest anti-obesity effect in the genetic rodent model of obesity. The relative importance of alpha2- and alpha1-adrenoceptors and imidazoline I2b-binding sites in mediating the effects of MPV-1743 A III needs further evaluation.

The effects of a beta 3-adrenoceptor agonist on insulin-induced changes in lipid metabolism were studied in obese male Zucker (fa/fa) rats during euglycaemic clamp. Rats were treated with BRL 35135 (R*, R*-(+/-)-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)-ethyl-amino]-propyl] phenoxyacetate hydrobromide) (0.5 mg/kg per day in drinking water) for three weeks before an euglycaemic hyperinsulinaemic clamp was performed. Insulin infusion lowered serum non-esterified fatty acids and plasma glycerol more efficiently in BRL 35135-treated than in control rats although plasma insulin remained significantly lower in the BRL 35135-treated than in the control rats during the clamp. In conclusion, chronic treatment with BRL 35135 potentiates the effect of insulin on lipid metabolism.

Aim Nutrition contributes to increased adiposity, but it remains to be determined whether high fat rather than Western diet exacerbates the development of obesity and other characteristics of metabolic syndrome and vascular function. Methods We studied the effects of high fat (45% kcal) diet (HFD) and equal caloric Western diet (WD) high in fat, sucrose and cholesterol for 8 weeks in male C57B1/6N mice. Results Mice fed with HFD and WD showed substantially higher body adiposity (body fat %) compared with control mice receiving low fat (10%) diet (LFD). However, total body weight was higher only in HFD mice compared with other groups. The amount of liver triglycerides, cholesterol and oxidative damage was higher in WD mice compared with mice on LFD. There were no significant differences in fasting blood glucose or serum insulin, serum or muscle triglycerides, glucose tolerance or systolic blood pressure between the groups, but serum free fatty acids were increased in HFD mice compared with LFD. Increased levels of tissue and serum diene conjugation as a marker of oxidative stress were evident especially in WD mice. The endothelium-dependent relaxations were significantly impaired in the small mesenteric arteries of HFD mice, but not in the aorta. Maximal relaxations correlated negatively with body adiposity in WD but not in HFD mice. Conclusions The major finding in the present study is that without changing body weight, Western diet induces marked whole-body oxidative stress and elevates body adiposity, which associates with the endothelial function of resistance arteries.

Insulin secretion is controlled by pancreatic α(2A)-adrenoceptors. Mice lacking α(2A)-adrenoceptors (α(2A)AR(-/-) mice) show hyperinsulinaemia, reduced blood glucose levels and improved glucose tolerance.In the present study, we used α(2AC)AR(-/-), α(2C)AR(-/-) and α(2A)AR(-/-) mice and a mouse line with adrenergic cell-specific expression of α(2A)-adrenoceptors (lacking these receptors in non-adrenergic cells), and their wild-type (WT) controls, to assess the glucoregulatory role of the α(2C)-adrenoceptor subtype in vivo. Glucose and insulin tolerance tests were performed and blood glucose and serum insulin levels were determined after fasting and glucose stimulation. Plasma catecholamines were also measured. In addition, the effect of pretreatment with (±)-propranolol was determined in α(2C)AR(-/-) mice.α(2AC)AR(-/-) mice had a similar glucose and insulin phenotype as α(2A)AR(-/-) mice and mice with restored α(2A)-autoreceptors, suggesting that only deletion of postsynaptic α(2A)-adrenoceptors has major effects on glucose disposition. However, α(2AC)AR(-/-) mice were more sensitive to the glucose-lowering effect of insulin than WT mice. This was not observed in α(2A)AR(-/-) mice. The α(2C)AR(-/-) mice showed impaired glucose tolerance that was reversed by pretreatment with (±)-propranolol. No difference in insulin secretion was observed in α(2C)AR(-/-) mice compared with WT animals.The results underline that depletion of postsynaptic pancreatic α(2A)-adrenoceptors has major effects on the regulation of glucose homeostasis in α(2AC)AR(-/-) and α(2A)AR(-/-) mice. Deletion of the α(2C) subtype leads to increased adrenaline secretion and has the potential to increase blood glucose levels via enhanced glycogenolysis.

Background and Aims: Neuropeptide Y (NPY) is a sympathetic neurotransmitter co-stored and co-released with noradrenaline and adrenaline. We have constructed a novel NPY transgenic mouse model (OE-NPYDBH mouse) where targeted overexpression results in increased levels of NPY in the brainstem and adrenal glands. The present study was aimed to understand the role of NPY released from sympathetic nerves and brain noradrenergic neurons in regulation of blood pressure, and behavioral responses to stress. Methods: Blood pressure was measured by radiotelemetry in conscious male OE-NPYDBH and wild-type mice during surgical stress and in baseline conditions. Plasma and adrenal gland catecholamine levels were measured at baseline. Acute immobilization and cold exposure were used to study the plasma levels of NPY and corticosterone in stress, and brown adipose tissue thermogenic activity was measured with [3H]GDP binding after cold. Results: Here, we demonstrate that sympathoadrenal activity is enhanced in the OE-NPYDBH mice. Blood pressure during surgical stress was significantly increased in comparison with wild-type controls. Furthermore, OE-NPYDBH mice showed sexually dimorphic NPY responses to stress, and an anxiolytic-like behavior in elevated plus-maze and light-dark tests. Conclusion: This study shows that the overactive noradrenergic NPY system plays a role in regulation of blood pressure and adaptive responses to stress, and may be a link between chronic stress and adiposity-associated disturbances in metabolism.

A functional polymorphism leucine 7 proline in the human neuropeptide Y (NPY) gene leading to increased NPY release from sympathetic nerves is associated with traits of metabolic syndrome. Although hypothalamic NPY neurons play an established role in promoting positive energy balance, the role of NPY colocalized with norepinephrine in sympathetic nervous system and brain noradrenergic neurons remains obscure.To clarify the role of NPY in noradrenergic neurons, we generated a transgenic mouse overexpressing NPY under dopamine-beta-hydroxylase promoter and characterized the metabolic phenotype of the OE-NPY(DbetaH) mouse.NPY levels are increased by 1.3-fold in adrenal glands and 1.8-fold in the brainstem but not in the hypothalamus in OE-NPY(DbetaH) mice. They display increased white adipose tissue mass and cellularity and liver triglyceride accumulation without hyperphagia or increased body weight. Hyperinsulinemia and impaired glucose tolerance develop by the age of 6 months in the OE-NPY(DbetaH) mice. Furthermore, circulating ghrelin is significantly increased in comparison with wild-type mice.The present study shows that even a moderate increase in NPY levels in noradrenergic neurons leads to disturbances in glucose and lipid metabolism. The OE-NPY(DbetaH) mouse is an interesting new model to investigate the pathophysiology of some key components of the cluster of abnormalities characterizing the metabolic syndrome.

Transgenic (TG) female mice, expressing a chimeric bovine luteinizing hormone (LH) β-subunit/human chorionic gonadotropin β-subunit COOH-terminal extension (bLHβ-CTP) gene, produce high levels of circulating LH and serve as a model for functional ovarian hyperandrogenism and follicular cysts. We report here that obesity is a typical feature of these female mice. The mean body weight of the bLHβ-CTP females was significantly higher than in controls at, and beyond 5 wk of age, and at 5 mo, it was 32% increased. At this age, the amount of white adipose tissue in the bLHβ-CTP females was significantly increased, as reflected by the weight difference of the retroperitoneal fat pad. In addition, the expression of leptin mRNA in white adipose tissue of the TG females was elevated about twofold. Serum leptin and insulin levels, and food intake, were also increased significantly in the TG females. Brown adipose tissue (BAT) thermogenic activity, as measured by GDP binding to BAT mitochondria, was reduced ( P < 0.05). Ovariectomy at the age of 3 wk totally prevented the development of obesity. In summary, the present results show that intact female bLHβ-CTP mice are obese, have increased food consumption, and reduced BAT thermogenic activity. The weight gain can be explained partly by elevated androgens but is probably also contributed to the increased adrenal steroidogeneisis. Hence, the bLHβ-CTP mice provide a useful model for studying obesity related to elevated LH secretion, with consequent alterations in ovarian and adrenal function.

Objective: Entacapone is a peripherally acting catechol O-methyltransferase (COMT) inhibitor used as an adjunct to each daily levodopa/dopa decarboxylase (DDC) inhibitor dose in the treatment of Parkinson's disease. Parkinsonian patients with advanced disease and motor fluctuations take several doses of levodopa daily, due to the short action of levodopa in this patient population. The present study was conducted in order to evaluate the pharmacokinetics of entacapone after multiple dosing and the pattern of COMT inhibition in erythrocytes during the first day of dosing as well as during steady state. Furthermore, the disposition of plasma levodopa and carbidopa was studied after a single dose of levodopa/carbidopa during the same conditions. Methods: Twelve healthy male volunteers received 200 mg entacapone eight times daily during study day 1 and day 6 at 2-h intervals from 0800 hours to 2200 hours. During days 3, 4 and 5, 200 mg of entacapone was taken ten times daily, from 0800 hours to 0200 hours on the following day. One levodopa/carbidopa tablet (100/25 mg) was taken on study day 1 and day 6 at 1000 hours. Plasma entacapone concentrations and erythrocyte COMT activities were measured frequently on study days 1–2 and 6–7, and twice daily on study days 3–5. Pharmacokinetic parameters calculated from plasma drug concentrations on days 1–2 and 6–7 were compared with each other. Results: There were no differences in maximal plasma concentration (Cmax), time to maximal drug concentration in plasma (tmax), elimination half-life (t1/2) and area under the plasma concentration–time curve (AUC) of entacapone between day 1 and day 6. The mean t1/2 values of entacapone were 1.3 h and 1.8 h during the first and sixth days, respectively; the difference was not significant. No signs of accumulation of entacapone were noted after the first day. Entacapone reduced erythrocyte COMT activity after the first dose, and this effect was quite stable during frequent dosing. There were no indications of accumulation of COMT inhibition during frequent dosing of entacapone. There were no between-day differences in Cmax, t1/2 (2.4 h on days 1–2 and 2.3 h on days 6–7) or AUC of levodopa, whereas tmax occurred at 0.8 h on day 1 and at 1.2 h on day 6 (P = 0.03). There were no between-day differences in the pharmacokinetic parameters (Cmax, tmax and AUC) of carbidopa. Conclusion: Even when dosed frequently, there are neither indications of accumulation of entacapone nor of its COMT inhibiting activity.

Selective β 3 -adrenoceptor agonists increase energy expenditure by increasing non-shivering thermogenesis in brown adipose tissue. The aim of this study was to investigate how changes in energy balance affect energy intake and interaction of peripheral metabolic feedback signals with central neuroendocrine mechanisms participating in the control of body energy balance. Expression of preproneuropeptide Y (preproNPY) mRNA in the arcuate nucleus and preprocorticotropin-releasing factor (CRF) mRNA in the paraventricular nucleus were measured by in situ hybridisation technique after 1 day, 1 and 5 weeks of treatment with ZD7114 (( S )-4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]- N -(2-methoxyethyl)phenoxyacetamide, 3 mg kg −1 day −1 in drinking water) in obese fa / fa Zucker rats. In addition, expression of leptin mRNA in epididymal fat and serum levels of leptin were analysed. Food intake, body weights, binding of GDP to brown adipose tissue mitochondria, plasma insulin and glucose were also measured. Treatment with ZD7114 significantly reduced weight gain and activated brown adipose tissue thermogenesis, but had no effect on food intake. Expressions of preproNPY or preproCRF mRNAs were similarly not changed by treatment with ZD7114. Furthermore, ZD7114 had no effect on plasma insulin or leptin and the expression of leptin mRNA in epididymal fat. However, statistically significant correlations were found between preproNPY and preproCRF mRNA expressions and brown fat thermogenic activity and plasma insulin levels in the ZD7114 treated rats, but not in the control rats. It is concluded that treatment with ZD7114 markedly activated brown fat thermogenesis, but did not affect neuropeptide Y (NPY) and CRF gene expression per se. However, the correlation analyses suggest that ZD7114 may modulate feedback connections of brown adipose tissue thermogenesis and plasma insulin with the hypothalamic neuroendocrine mechanisms integrating body energy balance.

Melanocyte-stimulating hormones, α-, β- and γ-MSH, regulate important physiological functions including energy homeostasis, inflammation and sodium metabolism. Previous studies have shown that α-MSH increases sodium excretion and promotes vascular function in rodents, but it is unexplored whether these characteristics of α-MSH could translate into therapeutic benefits in the treatment of hypertension. Therefore, we first assessed the diuretic and natriuretic properties of the stable α-MSH analogue [Nle(4), D-Phe(7)]-α-MSH (NDP-α-MSH) and investigated whether it has protective effects in deoxycorticosterone acetate (DOCA)-salt hypertensive mice. Adult male C57Bl/6N mice were subjected to DOCA-salt treatment and randomized to receive intraperitoneal injections of either saline as vehicle or NDP-α-MSH (0.3 mg/kg/day for 14 days) starting 7 days after the DOCA-salt treatment. Systemic hemodynamics, serum and urine electrolytes, and oxidative stress markers were assessed in control sham-operated and DOCA-salt mice. NDP-α-MSH elicited marked diuretic and natriuretic responses that were reversible with the MC3/4 receptor antagonist SHU9119. Chronic NDP-α-MSH treatment attenuated blood pressure elevation in DOCA-salt mice without affecting the blood pressure of normotensive control animals. Owing to the enhanced sodium excretion, NDP-α-MSH-treated mice were protected from DOCA-salt-induced hypernatremia. DOCA-salt treatment mildly increased oxidative stress at the tissue level, but NDP-α-MSH had no significant effects on the oxidative stress markers. In conclusion, treatment with NDP-α-MSH increases urinary sodium excretion and protects against DOCA-salt-induced hypertension. These findings point to the potential future use of α-MSH analogues in the treatment of hypertension.

Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13(-/-)) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13(-/-) mice. Granulation tissue in Mmp13(-/-) mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13(-/-) mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13(-/-) mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13(-/-) granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13(-/-) mice compared to WT mice. Mmp13(-/-) mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.

This study used two mouse models with genetic manipulation of the melanocortin system to investigate prolactin regulation. Mice with overexpression of the melanocortin receptor (MC-R) agonist, α-melanocyte-stimulating hormone (Tg-MSH) or deletion of the MC-R antagonist agouti-related protein (AgRP KO) were studied. Male Tg-MSH mice had lower blood prolactin levels at baseline (2.9±0.3 vs 4.7±0.7 ng/ml) and after restraint stress(68 ±6.5 vs 117±22 ng/ml) versus WT (p

Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede β-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes., Author Summary We have recently found that distinct metabolic disturbances precede β-cell autoimmunity in children who later progress to type 1 diabetes (T1D). Here we performed a murine study using non-obese diabetic (NOD) mice that recapitulated the protocol used in human, followed up by independent studies where NOD mice were studied in relation to risk of diabetes progression. We found that young female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum subgroup. The metabolic phenotypes observed in our study could be relevant as end points for studies investigating T1D pathogenesis and/or responses to interventions. By proceeding from a clinical study via metabolomics and modeling to an experimental model using a similar study design, then evolving further to tissue-specific studies, we hereby also present a conceptually novel approach to reversed translation that may be useful in future therapeutic studies in the context of prevention and treatment of T1D as well as of other diseases characterized by long prodromal periods.