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Aims/hypothesis Despite a similar fat storing function, visceral (intra-abdominal) white adipose tissue (WAT) is detrimental, whereas subcutaneous WAT is considered to protect against metabolic disease. Recent findings indicate that thermogenic genes, expressed in brown adipose tissue (BAT), can be induced primarily in subcutaneous WAT. Here, we investigate the hypothesis that the Wilms tumour gene product (WT1), which is expressed in intra-abdominal WAT but not in subcutaneous WAT and BAT, suppresses a thermogenic program in white fat cells. Methods Heterozygous Wt1 knockout mice and their wild-type littermates were examined in terms of thermogenic and adipocyte-selective gene expression. Glucose tolerance and hepatic lipid accumulation in these mice were assessed under normal chow and high-fat diet conditions. Pre-adipocytes isolated from the stromal vascular fraction of BAT were transduced with Wt1-expressing retrovirus, induced to differentiate and analysed for the expression of thermogenic and adipocyte-selective genes. Results Expression of the thermogenic genes Cpt1b and Tmem26 was enhanced and transcript levels of Ucp1 were on average more than tenfold higher in epididymal WAT of heterozygous Wt1 knockout mice compared with wild-type mice. Wt1 heterozygosity reduced epididymal WAT mass, improved whole-body glucose tolerance and alleviated severe hepatic steatosis upon diet-induced obesity in mice. Retroviral expression of WT1 in brown pre-adipocytes, which lack endogenous WT1, reduced mRNA levels of Ucp1, Ppargc1a, Cidea, Prdm16 and Cpt1b upon in vitro differentiation by 60–90%. WT1 knockdown in epididymal pre-adipocytes significantly lowered Aldh1a1 and Zfp423 transcripts, two key suppressors of the thermogenic program. Conversely, Aldh1a1 and Zfp423 mRNA levels were increased approximately five- and threefold, respectively, by retroviral expression of WT1 in brown pre-adipocytes. Conclusion/interpretation WT1 functions as a white adipocyte determination factor in epididymal WAT by suppressing thermogenic genes. Reducing Wt1 expression in this and other intra-abdominal fat depots may represent a novel treatment strategy in metabolic disease. Graphical abstract

Aims Heart failure (HF) is characterized by an overactivation of β-adrenergic signaling that directly contributes to impairment of myocardial function. Moreover, β-adrenergic overactivation induces adipose tissue lipolysis, which may further worsen the development of HF. Recently we demonstrated that adipose tissue-specific deletion of adipose triglyceride lipase (ATGL) prevents pressure-mediated HF in mice. In this study, we investigated the cardioprotective effects of a new pharmacological inhibitor of ATGL, Atglistatin, predominantly targeting ATGL in adipose tissue, on catecholamine-induced cardiac damage. Methods and results Male 129/Sv mice received repeated injections of isoproterenol (ISO, 25 mg/kg BW) to induce cardiac damage. Five days prior to ISO application, oral Atglistatin (2 mmol/kg diet) or control treatment was started. Two and twelve days after the last ISO injection cardiac function was analyzed by echocardiography. The myocardial deformation was evaluated using speckle-tracking-technique. Twelve days after the last ISO injection, echocardiographic analysis revealed a markedly impaired global longitudinal strain, which was significantly improved by application of Atglistatin. No changes of ejection fraction were observed. Further studies included histological-, WB-, and RT-qPCR-based analysis of cardiac tissue, followed by cell culture experiments and mass spectrometry-based lipidome analysis. ISO application induced subendocardial fibrosis and a profound pro-apoptotic cardiac response, as demonstrated using an apoptosis-specific gene expression-array. Atglistatin treatment led to a dramatic reduction of these pro-fibrotic and pro-apoptotic processes. We then identified a specific set of fatty acids (FAs) liberated from adipocytes under ISO stimulation (palmitic acid, palmitoleic acid and oleic acid), which induced pro-apoptotic effects in cardiomyocytes. Atglistatin significantly blocked this adipocytic FA secretion. Conclusions The present study demonstrates cardioprotective effects of Atglistatin in a mouse model of catecholamine-induced cardiac damage/dysfunction, involving anti-apoptotic and anti-fibrotic actions. Notably, beneficial cardioprotective effects of Atglistatin are likely mediated by non-cardiac actions, supporting the concept that pharmacological targeting of adipose tissue may provide an effective way to treat cardiac dysfunction. Translational perspective The pharmacological inhibition of ATGL activity in adipose tissue improves heart function in a murine model of catecholamine-induced myocardial damage, via significant reduction of cardiac apoptosis and fibrosis. Our data strongly support the role of an adipose tissue-heart communication in the development of cardiac diseases, associated with increased sympathetic-tone. Atglistatin beneficial actions were only mild, when applied after the catecholamine-induced damage in a therapeutic manner. However, when given prior to the event in a preventive manner, Atglistatin strongly protected against cardiac damage. These data suggest that an Atglistatin-based therapy may be more suitable as a new pharmacological option in cardiovascular prevention.

The effects of the selective AT 2 R (angiotensin AT2 receptor) agonist, Compound 21 (C21), on abdominal aortic aneurysm formation were investigated in normotensive Wistar rats. Abdominal aortic aneurysm was induced by perfusion of isolated aortic segments with elastase. Treatment with C21 (0.03 mg/kg daily) was started after operation and continued for 14 days. Sham-operated animals and vehicle-treated animals after aneurysm induction served as controls. Aortic diameter, aortic wall distensibility, and pulse propagation velocity were measured via ultrasound. Hemodynamic parameters, aortic tissue protein expression, and serum cytokines were analyzed. On day 14 post aneurysm induction, aortic diameter of vehicle-treated animals was increased 1.6-fold compared with sham-operated rats (2.65±0.05 versus 1.70±0.06 mm; P P P P 2 R stimulation with C21 prevented extracellular matrix degradation, maintained vascular integrity of the aorta and prevented abdominal aortic aneurysm progression.

Monocyte migration is a key element in atherosclerosis. LDL-C facilitates monocyte migration via induction of CCR2. PCSK9 regulates cell surface expression of the LDL-R and is expressed in vascular smooth muscle cells (VSMCs). The present study was done to investigate the regulation of PCSK9 in VSMCs and its impact on monocyte function. Methods and results PCSK9 mRNA and protein levels were upregulated in VSMCs by the TLR-4 ligand LPS, whereas TGF-β or angiotensin II had no effect. Induction of PCSK9 was selectively inhibited by TLR-4 blockade and further downstream by the SAPK/JNK-inhibitor SP600125, whereas inhibitors of ERK1/2, p38 or PI3-kinase pathways had no effect. Incubation of monocytes in conditioned media from LPS-stimulated VSMCs resulted in a significant reduction of LDL-R levels on monocytes, comparable to the effects of recombinant PCSK9. LDL-C increased monocyte CCR2 expression, which augmented monocyte migration towards MCP-1. This LDL-C dependent monocyte chemotaxis was inhibited by supernatants from LPS-stimulated VSMCs, similar to recombinant PCSK9 and a specific LDL-R blocking antibody. Conclusion PCSK9 is regulated in VSMCs by TLR-4 – SAPK/JNK signaling, a pathway important in inflammation and metabolism. VSMC-derived PCSK9 reduces monocyte LDL-R expression, affecting LDL-C/LDL-R-mediated CCR2-expression on monocytes, which is crucial to cell motility and atherogenesis.

Background Insulin resistance in visceral adipose tissue (VAT), skeletal muscle and liver is a prominent feature of most patients with obesity. How this association arises remains poorly understood. The objective of this study was to demonstrate that the decrease in insulin receptor (INSR) expression and insulin signaling in VAT from obese individuals is an early molecular manifestation that might play a crucial role in the cascade of events leading to systemic insulin resistance. Methods To clarify the role of INSR and insulin signaling in adipose tissue dysfunction in obesity, we first measured INSR expression in VAT samples from normal-weight subjects and patients with different degrees of obesity. We complemented these studies with experiments on high-fat diet (HFD)-induced obese mice, and in human and murine adipocyte cultures, in both normoxic and hypoxic conditions. Findings An inverse correlation was observed between increasing body mass index and decreasing INSR expression in VAT of obese humans. Our results indicate that VAT-specific downregulation of INSR is an early event in obesity-related adipose cell dysfunction, which increases systemic insulin resistance in both obese humans and mice. We also provide evidence that obesity-related hypoxia in VAT plays a determinant role in this scenario by decreasing INSR mRNA stability. This decreased stability is through the activation of a miRNA (miR-128) that downregulates INSR expression in adipocytes. Interpretation We present a novel pathogenic mechanism of reduced INSR expression and insulin signaling in adipocytes. Our data provide a new explanation linking obesity with systemic insulin resistance. Funding This work was partly supported by a grant from Nutramed (PON 03PE000_78_1) and by the European Commission (FESR FSE 2014-2020 and Regione Calabria).

Background: Obesity is a risk factor for diseases including type 2 diabetes mellitus (T2DM) and cardiovascular disorders. Diabetes itself contributes to cardiac damage. Thus, studying cardiovascular events and establishing therapeutic intervention in the period of type T2DM onset and manifestation are of highest importance. Mitochondrial dysfunction is one of the pathophysiological mechanisms leading to impaired cardiac function. Methods: An adequate animal model for studying pathophysiology of T2DM is the New Zealand Obese (NZO) mouse. These mice were maintained on a high-fat diet (HFD) without carbohydrates for 13 weeks followed by 4 week HFD with carbohydrates. NZO mice developed severe obesity and only male mice developed manifest T2DM. We determined cardiac phenotypes and mitochondrial function as well as cardiomyocyte signaling in this model. Results: The development of an obese phenotype and T2DM in male mice was accompanied by an impaired systolic function as judged by echocardiography and MyH6/7 expression. Moreover, the mitochondrial function only in male NZO hearts was significantly reduced and ERK1/2 and AMPK protein levels were altered. Conclusions: This is the first report demonstrating that the cardiac phenotype in male diabetic NZO mice is associated with impaired cardiac energy function and signaling events.

Sterile inflammation of visceral fat, provoked by dying adipocytes, links the metabolic syndrome to cardiovascular disease. Danger-associated molecular patterns, such as adenosine triphosphate (ATP), are released by activated or dying cells and orchestrate leukocyte infiltration and inflammation via the purinergic receptor P2Y2. The gene expression of ATP receptor P2Y2 did not change in several tissues in the course of obesity, but was increased within epididymal fat. Adipose tissue from P2Y 2 −/− mice consuming high-fat diet (HFD) contained less crown-like structures with a reduced frequency of adipose tissue macrophages (ATMs). This was likely due to decreased leukocyte migration because of missing VCAM-1 exposition on P2Y2 deficient hypertrophic adipose tissue endothelial cells. Accordingly, P2Y 2 −/− mice showed blunted traits of the metabolic syndrome: they gained less weight compared to P2Y 2 +/+ controls, while intake of food and movement behaviour remained unchanged. Liver and adipose tissue were smaller in P2Y 2 −/− animals. Insulin tolerance testing (ITT) performed in obese P2Y 2 −/− mice revealed a better insulin sensitivity as well as lower plasma C-peptide and cholesterol levels. We demonstrate that interfering with somatic P2Y2 signalling prevents excessive immune cell deposition in diet-induced obesity (DIO), both attenuating adipose tissue inflammation and ameliorating the metabolic phenotype. Thus, blocking the P2Y2 cascade may be a promising strategy to limit metabolic disease and its sequelae.

Rationale: The coincidence of inflammation and metabolic derangements in obese adipose tissue has sparked the concept of met-inflammation. Previous observations, however, suggest that inflammatory pathways may not ultimately cause dysmetabolism. Objective: We have revisited the relationship between inflammation and metabolism by testing the role of TRAF (tumor necrosis receptor-associated factor)-1, an inhibitory adapter of inflammatory signaling of TNF (tumor necrosis factor)-a, IL (interleukin)-1ß, and TLRs (toll-like receptors). Methods and Results: Mice defcient for TRAF-1, which is expressed in obese adipocytes and adipose tissue lymphocytes, caused an expected hyperinflammatory phenotype in adipose tissue with enhanced adipokine and chemokine expression, increased leukocyte accumulation, and potentiated proinflammatory signaling in macrophages and adipocytes in a mouse model of diet-induced obesity. Unexpectedly, TRAF-1-/-mice were protected from metabolic derangements and adipocyte growth, failed to gain weight, and showed improved insulin resistance-an effect caused by increased lipid breakdown in adipocytes and UCP (uncoupling protein)-1-enabled thermogenesis. TRAF-1-dependent catabolic and proinflammatory cues were synergistically driven by ß3-adrenergic and inflammatory signaling and required the presence of both TRAF-1-defcient adipocytes and macrophages. In human obesity, TRAF-1-dependent genes were upregulated. Conclusions: Enhancing TRAF-1-dependent inflammatory pathways in a gain-of-function approach protected from metabolic derangements in diet-induced obesity. These fndings identify TRAF-1 as a regulator of dysmetabolism in mice and humans and question the pathogenic role of chronic inflammation in metabolism. Fil: Michel, Nathaly Anto. University of Freiburg. University Medical Center; Alemania Fil: Colberg, Christian. University of Freiburg. University Medical Center; Alemania Fil: Buscher, Konrad. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Sommer, Björn. Universitat Erlangen-Nuremberg; Alemania Fil: Pramod, Akula Bala. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Ehinger, Erik. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Dufner, Bianca. University of Freiburg. University Medical Center; Alemania Fil: Hoppe, Natalie. University of Freiburg. University Medical Center; Alemania Fil: Pfeiffer, Katharina. University of Freiburg. University Medical Center; Alemania Fil: Marchini, Timoteo Oscar. University of Freiburg. University Medical Center; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Willecke, Florian. University of Freiburg. University Medical Center; Alemania Fil: Stachon, Peter. University of Freiburg. University Medical Center; Alemania Fil: Hilgendorf, Ingo. University of Freiburg. University Medical Center; Alemania Fil: Heidt, Timo. University of Freiburg. University Medical Center; Alemania Fil: Von Zur Muhlen, Constantin. University of Freiburg. University Medical Center; Alemania Fil: Von Elverfeldt, Dominik. University of Freiburg. University Medical Center; Alemania Fil: Pfeifer, Dietmar. University of Freiburg; Alemania Fil: Schüle, Roland. University of Freiburg; Alemania Fil: Kintscher, Ulrich. University of Freiburg; Alemania Fil: Brachs, Sebastian. Center For Cardiovascular Research; Alemania Fil: Ley, Klaus. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Bode, Christoph. University of Freiburg. University Medical Center; Alemania Fil: Zirlik, Andreas. University of Freiburg. University Medical Center; Alemania Fil: Wolf, Dennis. La Jolla Institute for Allergy and Immunology; Estados Unidos

Objective: Insulin resistance can be triggered by enhanced dephosphorylation of the insulin receptor or downstream components in the insulin signaling cascade through protein tyrosine phosphatases (PTPs). Downregulating density-enhanced phosphatase-1 (DEP-1) resulted in an improved metabolic status in previous analyses. This phenotype was primarily caused by hepatic DEP-1 reduction. Methods: Here we further elucidated the role of DEP-1 in glucose homeostasis by employing a conventional knockout model to explore the specific contribution of DEP-1 in metabolic tissues. Ptprj−/− (DEP-1 deficient) and wild-type C57BL/6 mice were fed a low-fat or high-fat diet. Metabolic phenotyping was combined with analyses of phosphorylation patterns of insulin signaling components. Additionally, experiments with skeletal muscle cells and muscle tissue were performed to assess the role of DEP-1 for glucose uptake. Results: High-fat diet fed-Ptprj−/− mice displayed enhanced insulin sensitivity and improved glucose tolerance. Furthermore, leptin levels and blood pressure were reduced in Ptprj−/− mice. DEP-1 deficiency resulted in increased phosphorylation of components of the insulin signaling cascade in liver, skeletal muscle and adipose tissue after insulin challenge. The beneficial effect on glucose homeostasis in vivo was corroborated by increased glucose uptake in skeletal muscle cells in which DEP-1 was downregulated, and in skeletal muscle of Ptprj−/− mice. Conclusion: Together, these data establish DEP-1 as novel negative regulator of insulin signaling.

The renin-angiotensin system (RAS) and obesity have been implicated in vascular outward remodeling, including aneurysms, but the precise mechanisms are not yet understood. We investigated the effect of the angiotensin receptor type 1 (AT1-receptor) antagonist telmisartan on aortic outward remodeling in a diet-induced obesity model in mice. C57/Black6J mice were fed either a low-fat diet (LFD) or a high-fat diet (HFD) for 14 weeks. One group of HFD mice was additionally exposed to telmisartan (3mg/kg per day) for the last 4 weeks. HFD led to aortic outward remodeling, characterized by increased proteolysis, along with structural changes, such as fragmentation of elastic fibers and decreased elastin content. Vascular damage was associated with up-regulation of matrix metalloproteinase (MMP)-2 (MMP-2), MMP-3, MMP-12, cathepsin D, and cathepsin B. HFD aortae exhibited an enhanced inflammatory status, characterized by tumor necrosis factor alpha(TNF-alpha) and interleukin-1 beta (IL-1 beta) colocalized with adipocytes in the adventitia. HFD resulted in a significant increase in aortic dimensions, evident by ultrasound measurements. Telmisartan abolished aortic dilatation and preserved elastin content. HFD induced enhanced expression of aortic MMP-2, MMP-9, and TNF-alpha was abrogated by telmisartan. Adventitial proteolytic and inflammatory factors were also examined in samples from human abdominal aneurysms. The expression of TNF-alpha, IL-1 beta, and MMP-9 was higher in the adventitial fat of diseased vessels compared with healthy tissues. Finally, adipocytes treated with TNF-alpha showed enhanced MMP-2, MMP-3, and cathepsin D, which was prevented by telmisartan. Taken together, HFD in mice induced aortic dilatation with up-regulation of matrix degrading and inflammatory pathways similar to those seen in human aortic aneurysmatic tissue. The HFD-induced vascular pathology was reduced by AT1-receptor antagonist telmisartan.

Evidence for a critical pathophysiological role of aberrant cytoskeletal dynamics is being uncovered in a growing number of neuropsychiatric syndromes. A sedentary lifestyle as well as overt psychopathology is prevalent in patients with the metabolic syndrome. Using mice deficient in gelsolin (Gsn-/-), a crucial actin-severing protein, we here investigated reduced actin turnover as a potential common driver of metabolic disturbances, sedentary behavior, and an anxious/depressive phenotype. Gelsolin deficiency resulted in reduced lifespan. As compared to wildtype controls, Gsn-/- mice (~ 9 weeks) fed a high-fat diet (HFD) over a span of 12 weeks showed increased body weight gain, fat mass, hepatic steatosis, and adipocyte hypertrophy as well as a significantly reduced respiratory quotient. Moreover, increased rigidity of the actin cytoskeleton in mice on HFD induced mRNA expression of Acc1, Acc2, Fasn, and Lipe, key genes involved in fatty acid metabolism in the liver. Glucose tolerance and insulin sensitivity were worsened in Gsn-/- HFD relative to Gsn+/+ HFD mice. Hypertension in Gsn-/- mice was associated with reduced endothelial NO synthase (eNOS) mRNA expression and reduced eNOS protein trafficking to the plasma membrane. Furthermore, acetylcholine-induced cGMP production and relaxation of aortic rings were impaired by actin filament stabilization. Gsn-/- mice on HFD displayed reduced corticosterone concentrations and reduced energy expenditure as compared to Gsn+/+ HFD mice. Moreover, Gsn-/- HFD mice displayed an overall pattern of hypoactive and anxious/depressive-like behavior. In aggregate, our results demonstrate that impaired actin filament dynamics promote the development of key behavioral and physiological aspects of the metabolic syndrome.

Estrogen receptor alpha (ERα) is a major regulator of metabolic processes in obesity. In this study we aimed to define the relevance of adipose tissue ERα during high-fat diet (HFD)-induced obesity using female aP2-Cre−/+/ERαfl/fl mice (atERαKO). HFD did not affect body weight or glucose metabolism in atERαKO- compared to control mice. Surprisingly, HFD feeding markedly increased mortality in atERαKO mice associated with a destructive bacterial infection of the uterus driven by commensal microbes, an alteration likely explaining the absence of a metabolic phenotype in HFD-fed atERαKO mice. In order to identify a mechanism of the exaggerated uterine infection in HFD-fed atERαKO mice, a marked reduction of uterine M2-macrophages was detected, a cell type relevant for anti-microbial defence. In parallel, atERαKO mice exhibited elevated circulating estradiol (E2) acting on E2-responsive tissue/cells such as macrophages. Accompanying cell culture experiments showed that despite E2 co-administration stearic acid (C18:0), a fatty acid elevated in plasma from HFD-fed atERαKO mice, blocks M2-polarization, a process known to be enhanced by E2. In this study we demonstrate an unexpected phenotype in HFD-fed atERαKO involving severe uterine bacterial infections likely resulting from a previously unknown negative interference between dietary FAs and ERα-signaling during anti-microbial defence.

Left ventricular (LV) remodeling is the main reason for the development of progressive cardiac dysfunction after myocardial infarction (MI). This study investigated whether stimulation of the angiotensin type 2 receptor is able to ameliorate post-MI cardiac remodeling and what the underlying mechanisms may be. MI was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with the angiotensin type 2 receptor agonist compound 21 ( 0.03 mg/kg) was started 6 hours post-MI and continued for 6 weeks. Hemodynamic parameters were measured by echocardiography and intracardiac catheter. Effects on proteolysis were studied in heart tissue and primary cardiac fibroblasts. Compound 21 significantly improved systolic and diastolic functions, resulting in improved ejection fraction (71.2±4.7% versus 53.4±7.0%; P P P P P

In the present study we investigated the influence of sex difference on the development of left ventricular hypertrophy (LVH) during obesity. Male and female C57BL/6J mice were fed for 15 and 25 wk with a high-fat diet (HFD) or low-fat control diet (LFD). Analysis of body composition, monitoring of body weight (BW), and echocardiographic analysis were performed, as well as analysis of expression of different adipocytokines in epicardial adipose tissue. The increment in left ventricular mass (LVM) after HFD (25 wk) was significantly stronger in male mice compared with female mice [LVM: male, 116.9 ± 2.9 (LFD) vs. 142.2 ± 9.3 mg (HFD); female, 84.3 ± 3.3 (LFD) vs. 93.9 ± 1.7 mg (HFD), Psex < 0.01]. In parallel, males developed a higher BW and fat mass after 25 wk HFD than female mice [BW: male, 33 ± 0.9 (LFD) vs. 53 ± 0.8 g (HFD); fat mass: male, 8.8 ± 0.9 (LFD) vs. 22.8 ± 0.7 g (HFD); BW: female, 22.5 ± 0.4 (LFD) vs. 33.7 ± 1.3 g (HFD); fat mass: female, 4.0 ± 0.2 (LFD) vs. 13.2 ± 1.2 g (HFD)] ( P < 0.01 for BW+ fat mass female vs. male). The mRNA expression of adipocytokines in epicardial fat after 25 wk of diet showed higher levels of adiponectin (2.8-fold), leptin (4.2-fold), and vaspin (11.9-fold) in male mice compared with female mice ( P < 0.05). To identify new adipose-derived molecular mediators of LVH, we further elucidated the cardiac impact of vaspin. Murine primary cardiac fibroblast proliferation was significantly induced by vaspin (1.8-fold, vaspin 1 μg/l, P < 0.05 vs. control) compared with 1.9-fold induction by angiotensin II (10 μM). The present study demonstrates a sex-dependent regulation of diet-induced LVH associated with sexual dimorphic expression of adipocytokines in epicardial adipose tissue.

The cannabinoid receptors, CB1 and CB2, are expressed in the heart, but their role under pathological conditions remains controversial. This study examined the effect of CB1 receptor blockade on cardiovascular functions after experimental MI and in experimental metabolic syndrome. MI was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with the CB1 receptor antagonist rimonabant (10 mg/kg i.p. daily) started 7 days before or 6 h after MI and continued for 6 weeks. Haemodynamic parameters were measured via echocardiography and intracardiac Samba catheter. CB1 blockade improved systolic and diastolic heart function, decreased cardiac collagen and hydroxyproline content and down-regulated TGF-beta 1. Additionally, rimonabant decreased arterial stiffness, normalised QRS complex duration and reduced brain natriuretic peptide levels in serum. In primary cardiac fibroblasts, rimonabant decreased MMP-9 activity and TGF-beta 1 expression. Furthermore, rimonabant improved depressed systolic function of spontaneously hypertensive obese rats and reduced weight gain. Blocking of CB1 receptor with rimonabant improves cardiac functions in the early and late stages after MI, decreases arterial stiffness and reduces cardiac remodelling. Rimonabant also has cardioprotective actions in rats characterised by the metabolic syndrome. Inhibition of proteolysis and TGF-beta 1 expression and reduced collagen content by rimonabant may attenuate destruction of the extracellular matrix and decrease fibrosis after MI.

Different stimuli confer mechanical and hormonal stress to the heart. Pressure overload (PO) affects the myocardium, kidney, and peripheral fat. The latter may be considered simultaneously as a novel end organ and as a mediator in cardiovascular damage and, by its interplay with the heart, contributes to the development of pathological myocardial hypertrophy (MH). In contrast to pathological MH, in physiological MH the heart serves as an end organ for recurrent pressure spikes without the development of pathology. Complex stressors such as mineralocorticoid excess and salt challenge induce complex pathological cardiac and renal phenotypes. Understanding the underlying sex-specific mechanisms mediating these processes may help to design new therapeutic strategies. The major goal of this review is to analyze sex differences in physiological and pathological forms of cardiovascular stress and its effects on different organs, including the heart, kidney, and peripheral fat. Exercise-induced physiological MH differs markedly from pathological MH resulting from hypertension, valve disease, or other challenges. Physiological MH is reversible, associated with improved cardiac function and lack of fibrosis.1 At the level of cardiac metabolism, MH during the course of physical training is accompanied by enhanced fatty acid oxidation and reduced glucose use. ### Clinical Impact and Relevance in Humans Studies in humans comparing exercise-mediated MH in both sexes are limited. Left ventricular mass (LVM) is significantly increased in male endurance athletes by 36±14% compared with untrained control subjects.2 In young adult elite athletes, both sexes exhibited similar increases in LVM and LVM indices when compared with their sex-matched sedentary controls.3 However, limitations of these studies in humans have to be taken into account: despite a comparable training duration (hours/week and years at elite level) in female and male athletes, no detailed information about training intensity was provided.3 Putative sex differences in training intensity might have influenced exercise-induced cardiac remodeling. A lower …

Pharmacological blockade of mineralocorticoid receptors (MR) is known as an efficacious therapy in chronic heart failure. Therapy with steroidal MR antagonists such as spironolactone or eplerenone (EPL) is often limited because of side effects. Recently, a new highly selective and potent, nonsteroidal MR antagonist, finerenone (FIN), has been developed. To investigate the effects of FIN on pressure-induced cardiac hypertrophy, the transverse aortic constriction (TAC) model was used in C57BL/6 mice treated with FIN (10 mg·kg·d), EPL (200 mg·kg·d) or vehicle (VEH). First, we analyzed cardiac gene expression 4 weeks after TAC using a pathway-focused quantitative polymerase chain reaction array. FIN caused a distinct cardiac gene expression profile compared to VEH and EPL, including differential expression of BNP (brain natriuretic peptide) and Tnnt2 (troponin T type 2). FIN treatment led to a significant reduction of TAC-induced left ventricular (LV) wall thickening assessed by echocardiography. In accordance, FIN-treated mice showed a significant lower increase of calculated left ventricular mass compared with VEH- and EPL-treated mice (FIN: 28.4 ± 3.7 mg; EPL: 38.4 ± 4.3 mg; VEH: 39.3 ± 3.1 mg; P < 0.05). These data show beneficial effects of nonsteroidal MR antagonism by FIN on left ventricular mass development in pressure overload associated with a distinct cardiac gene expression profile.

Glucose-dependent insulinotropic polypeptide (GIP) has been suggested to have direct effects on nonislet tissues. GIP also reportedly increased glucose uptake and inhibition of lipolysis in adipocytes after inhibition of the intracellular cortisone-cortisol shuttle 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). We here analyzed whether GIP modifies lipid metabolism and further elucidated the relation between GIP, 11β-HSD1, and fatty acid metabolism. GIP reduced activity of 11β-HSD1 promoter constructs and the expression and activity of 11β-HSD1 in differentiated 3T3-L1 adipocytes in a time- and dose-dependent fashion. This was paralleled by a reduction of free fatty acid (FFA) release and a reduced expression of key enzymes regulating lipolysis in adipose tissue. Preinhibition of 11β-HSD1 completely abolished GIP-induced effects on FFA release. To investigate the acute effects of GIP in humans, a randomized clinical trial was performed. GIP lowered circulating FFAs compared with saline control and reduced expression and ex vivo activity of 11β-HSD1 and adipose triglyceride lipase expression in subcutaneous fat biopsies. Our data suggest that GIP reduces FFA release from adipose tissue by inhibition of lipolysis or by increased reesterification. This process appears to depend on a modification of 11β-HSD1 activity. In general, the presented data support that GIP has direct and insulin-independent effects on adipose tissue.

In the current study, we investigated the importance of histone deacetylase (HDAC)6 for glucocorticoid receptor–mediated effects on glucose metabolism and its potential as a therapeutic target for the prevention of glucocorticoid-induced diabetes. Dexamethasone-induced hepatic glucose output and glucocorticoid receptor translocation were analyzed in wild-type (wt) and HDAC6-deficient (HDAC6KO) mice. The effect of the specific HDAC6 inhibitor tubacin was analyzed in vitro. wt and HDAC6KO mice were subjected to 3 weeks’ dexamethasone treatment before analysis of glucose and insulin tolerance. HDAC6KO mice showed impaired dexamethasone-induced hepatic glucocorticoid receptor translocation. Accordingly, dexamethasone-induced expression of a large number of hepatic genes was significantly attenuated in mice lacking HDAC6 and by tubacin in vitro. Glucose output of primary hepatocytes from HDAC6KO mice was diminished. A significant improvement of dexamethasone-induced whole-body glucose intolerance as well as insulin resistance in HDAC6KO mice compared with wt littermates was observed. This study demonstrates that HDAC6 is an essential regulator of hepatic glucocorticoid-stimulated gluconeogenesis and impairment of whole-body glucose metabolism through modification of glucocorticoid receptor nuclear translocation. Selective pharmacological inhibition of HDAC6 may provide a future therapeutic option against the prodiabetogenic actions of glucocorticoids.

The present study aimed to explore the anti-inflammatory effects and peroxisome proliferator-activated receptor-γ (PPARγ)–activating properties of the angiotensin type 1 receptor blocker telmisartan by analysis of serum interleukin 6 levels and monocytic PPARγ target gene expression in drug-naïve patients with the metabolic syndrome. This was a 14-week, randomized, double-blind, placebo-controlled 2-center study with telmisartan 80 mg/d and telmisartan 160 mg/d in 54 patients with the metabolic syndrome. In addition to clinical laboratory measurements, peripheral monocytes were extracted by negative isolation using a Dynal Monocyte kit to evaluate ligand-activated PPARγ target gene expression (CD36 and CD163) at baseline and study end using quantitative real-time RT-PCR. In this low-risk patient population, telmisartan (80 and 160 mg) treatment did not significantly affect serum interleukin 6 levels. Expression of the PPARγ target gene CD36 in monocytes was markedly induced by telmisartan from baseline to study end (telmisartan 80 mg: 2.3±1.5-fold change versus placebo [ P value not significant]; telmisartan 160 mg: 3.5±0.9-fold change versus placebo [ P P value not significant]; telmisartan 160 mg: 1.4±0.4-fold change versus placebo [ P value not significant]), which did not reach statistical significance. This is the first clinical description of monocytic PPARγ target gene regulation with high-dose telmisartan treatment. These data implicate that the angiotensin type 1 receptor blocker telmisartan activates PPARγ in circulating monocytes of patients with the metabolic syndrome.

OBJECTIVE To determine the concentrations of adipocyte fatty acid–binding protein (AFABP) and other adipocytokines in maternal and cord serum of pregnant women with gestational diabetes mellitus (GDM) and of control subjects and to relate them to indexes of insulin sensitivity. RESEARCH DESIGN AND METHODS In 86 control and 98 GDM pregnant women, venous blood was collected before vaginal delivery and arterial blood from cord immediately after delivery. Serum insulin and adipocytokines were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS GDM women had higher prepregnancy BMI, and data were adjusted for it. Maternal serum insulin, insulin-to-glucose ratio, homeostasis model assessment (HOMA), AFABP, and retinol-binding protein 4 (RBP4) were higher and adiponectin was lower in GDM than in control subjects, whereas serum glucose, insulin, insulin-to-glucose ratio, HOMA, nonesterified fatty acids, and RBP4 were higher and glycerol, AFABP, and adiponectin were lower in cord blood serum of GDM than of control subjects. AFABP and adiponectin in cord serum of control subjects were higher than in maternal serum; in GDM women no difference was found for AFABP in cord versus maternal serum, although adiponectin remained higher in cord. Values of leptin in both groups were lower in cord than in maternal serum, and those of RBP4 were lower in only GDM women. CONCLUSIONS It is suggested that fetal tissues are the main source of cord arterial serum AFABP, and in GDM fetuses AFABP values correlate with adiposity markers. A downregulation of adiponectin and upregulation of RBP4 in GDM mothers and their fetuses may be related to their insulin-resistant condition, whereas changes in AFABP do not seem to be related.

Exercise-induced cardiac hypertrophy has been recently identified to be regulated in a sex-specific manner. In parallel, women exhibit enhanced exercise-mediated lipolysis compared with men, which might be linked to cardiac responses. The aim of the present study was to assess if previously reported sex-dependent differences in the cardiac hypertrophic response during exercise are associated with differences in cardiac energy substrate availability/utilization. Female and male C57BL/6J mice were challenged with active treadmill running for 1.5 h/day (0.25 m/s) over 4 wk. Mice underwent cardiac and metabolic phenotyping including echocardiography, small-animal PET, peri-exercise indirect calorimetry, and analysis of adipose tissue (AT) lipolysis and cardiac gene expression. Female mice exhibited increased cardiac hypertrophic responses to exercise compared with male mice, measured by echocardiography [percent increase in left ventricular mass (LVM): female: 22.2 ± 0.8%, male: 9.0 ± 0.2%; P < 0.05]. This was associated with increased plasma free fatty acid (FFA) levels and augmented AT lipolysis in female mice after training, whereas FFA levels from male mice decreased. The respiratory quotient during exercise was significantly lower in female mice indicative for preferential utilization of fatty acids. In parallel, myocardial glucose uptake was reduced in female mice after exercise, analyzed by PET {injection dose (ID)/LVM [%ID/g]: 36.8 ± 3.5 female sedentary vs. 28.3 ± 4.3 female training; P < 0.05}, whereas cardiac glucose uptake was unaltered after exercise in male counterparts. Cardiac genes involved in fatty acid uptake/oxidation in females were increased compared with male mice. Collectively, our data demonstrate that sex differences in exercise-induced cardiac hypertrophy are associated with changes in cardiac substrate availability and utilization.

Estrogens, acting on both estrogen receptors alpha (ERalpha) and beta (ERbeta) are recognized as important regulators of glucose homeostasis and lipid metabolism. ERs belong to the family of nuclear hormone receptors which mainly act as ligand activated transcription factors. Both ERs are expressed in metabolic tissue such as adipose tissue, skeletal muscle, liver and pancreas, as well as in the central nervous system. Expression pattern of both ERs differ between species, sexes, and specific tissues. The present review will focus on the key effects of ERs on glucose- and lipid metabolism. It appears that ERalpha mainly mediates beneficial metabolic effects of estrogens such as anti-lipogenesis, improvement of insulin sensitivity and glucose tolerance, and reduction of body weight/fat mass. In contrast, ERbeta activation seems to be detrimental for the maintenance of regular glucose and lipid homeostasis. Metabolic actions of both receptors in relevant tissues will be discussed.

The objective of the current study was to investigate the hypothesis that high-density lipoprotein (HDL) influences adipocyte metabolism and adiponectin expression. Therefore, HDL was increased in vivo via apolipoprotein (apo) A-I gene transfer and in vitro via supplementation of HDL to partly differentiated adipocytes, in the presence or absence of lipopolysaccharide (LPS), known to decrease HDL cholesterol and adiponectin levels in vivo.Apo A-I transfer resulted in a significant increase of HDL cholesterol in control and LPS-injected C57BL/6 mice, which was paralleled by an increase in plasma adiponectin levels and adiponectin expression in abdominal fat. Triglyceride and free fatty acids levels after LPS administration were 2.2-fold (p0.05) and 1.3-fold (p0.05) lower, respectively, in Ad.hapoA-I-LPS than in Ad.Null-LPS mice. In parallel, the LPS-induced mRNA expression of hormone sensitive lipase was 3.5-fold (p=0.05) decreased in the Ad.hapoA-I-LPS group. On the other hand, apo A-I transfer abrogated the LPS-mediated reduction in lipin-1 and CD36 mRNA expression by 8.2-fold (p0.05) and 18-fold (p0.05), respectively. Concomitantly, the phosphorylation state of Akt was 2.0-fold (p0.05) increased in the Ad.hapoA-I-LPS compared to the Ad.Null-LPS group. Pre-incubation of partly differentiated adipocytes with HDL (50 microg protein/ml) increased adiponectin expression by 1.5-fold under basal conditions (p0.05) and could abrogate LPS-induced down-regulation of adiponectin, both in a phosphatidylinositol-3-kinase-dependent manner.HDL affects adipocyte metabolism and adiponectin expression.

OBJECTIVE Fibroblast growth factor (FGF)-21 improves insulin sensitivity and lipid metabolism in obese or diabetic animal models, while human studies revealed increased FGF-21 levels in obesity and type 2 diabetes. Given that FGF-21 has been suggested to be a peroxisome proliferator–activator receptor (PPAR) α–dependent regulator of fasting metabolism, we hypothesized that free fatty acids (FFAs), natural agonists of PPARα, might modify FGF-21 levels. RESEARCH DESIGN AND METHODS The effect of fatty acids on FGF-21 was investigated in vitro in HepG2 cells. Within a randomized controlled trial, the effects of elevated FFAs were studied in 21 healthy subjects (13 women and 8 men). Within a clinical trial including 17 individuals, the effect of insulin was analyzed using an hyperinsulinemic-euglycemic clamp and the effect of PPARγ activation was studied subsequently in a rosiglitazone treatment trial over 8 weeks. RESULTS Oleate and linoleate increased FGF-21 expression and secretion in a PPARα-dependent fashion, as demonstrated by small-interfering RNA–induced PPARα knockdown, while palmitate had no effect. In vivo, lipid infusion induced an increase of circulating FGF-21 in humans, and a strong correlation between the change in FGF-21 levels and the change in FFAs was observed. An artificial hyperinsulinemia, which was induced to delineate the potential interaction between elevated FFAs and hyperinsulinemia, revealed that hyperinsulinemia also increased FGF-21 levels in vivo, while rosiglitazone treatment had no effect. CONCLUSIONS The results presented here offer a mechanism explaining the induction of the metabolic regulator FGF-21 in the fasting situation but also in type 2 diabetes and obesity.

Background— Adipose tissue inflammation may play a critical role in the pathogenesis of insulin resistance (IR). The present study examined the role of lymphocytes in adipose tissue inflammation and IR. Methods and Results— In a mouse model of obesity-mediated IR, high-fat diet (HFD) induced IR already after 5 weeks, which was associated with a marked T-lymphocyte infiltration in visceral adipose tissue. In contrast, recruitment of macrophages was delayed with an increase of MAC3-positive staining and F4/80 mRNA expression after 10 weeks of HFD, suggesting a dissociation of macrophage invasion into adipose tissue and IR initiation. In patients with type 2 diabetes, lymphocyte content in adipose tissue biopsies significantly correlated with waist circumference, a marker of IR. Immunohistochemical staining of human adipose tissue revealed the presence of mainly CD4-positive lymphocytes as well as macrophage infiltration. Most macrophages were HLA-DR–positive, reflecting activation through IFNγ, a cytokine released from CD4-positive lymphocytes. Conclusions— Proinflammatory T-lymphocytes are present in visceral adipose tissue and may contribute to local inflammatory cell activation before the appearance of macrophages, suggesting that these cells could play an important role in the initiation and perpetuation of adipose tissue inflammation as well as the development of IR.

OBJECTIVE—The angiotensin type 1 receptor blocker (ARB) and peroxisome proliferator–activated receptor (PPAR) γ modulator telmisartan has been recently demonstrated to reduce plasma triglycerides in nondiabetic and diabetic hypertensive patients. The present study investigates the molecular mechanisms of telmisartans hypolipidemic actions, in particular its effect on the PPARα pathway. RESEARCH DESIGN AND METHODS—Regulation of PPARα target genes by telmisartan was studied by real-time PCR and Western immunoblotting in vitro and in vivo in liver/skeletal muscle of mice with diet-induced obesity. Activation of the PPARα ligand binding domain (LBD) was investigated using transactivation assays. RESULTS—Telmisartan significantly induced the PPARα target genes carnitine palmitoyl transferase 1A (CPT1A) in human HepG2 cells and acyl-CoA synthetase long-chain family member 1 (ACSL1) in murine AML12 cells in the micromolar range. Telmisartan-induced CPT1A stimulation was markedly reduced after small interfering RNA–mediated knockdown of PPARα. Telmisartan consistently activated the PPARα-LBD as a partial PPARα agonist. Despite high in vitro concentrations required for PPARα activation, telmisartan (3 mg · kg−1 · day−1) potently increased ACSL1 and CPT1A expression in liver from diet-induced obese mice associated with a marked decrease of hepatic and serum triglycerides. Muscular CPT1B expression was not affected. Tissue specificity of telmisartan-induced PPARα target gene induction may be the result of previously reported high hepatic concentrations of telmisartan. CONCLUSIONS—The present study identifies the ARB/PPARγ modulator telmisartan as a partial PPARα agonist. As a result of its particular pharmacokinetic profile, PPARα activation by telmisartan seems to be restricted to the liver. Hepatic PPARα activation may provide an explanation for telmisartan's antidyslipidemic actions observed in recent clinical trials.

We tested the hypothesis that female and male mice differ in terms of cardiac hypertrophy after deoxycorticosterone acetate (DOCA)+salt hypertension (uninephrectomy and 1% saline in drinking water) and focused on calcineurin signaling. We excluded confounding effects of blood pressure elevation or sex-related blood pressure differences by treating DOCA-salt mice with hydralazine (250 mg/L in drinking water). We found that directly measured mean arterial blood pressure was lowered to control values with hydralazine and corroborated this finding in separate mouse groups with radiotelemetry. Male mice were more responsive to DOCA-salt–related effects. They developed more left ventricular hypertrophy and more renal hypertrophy after 6 weeks of DOCA-salt+hydralazine compared with female mice. In hearts, transcripts for calcineurin Aβ and for myocyte-enriched calcineurin interacting protein 1 were upregulated in male but not in female mice. Enhanced activity of calcineurin Aβ, as indicated by diminished phosphorylation of NFATc2 in male mice, accounted for this sex-specific difference. Stretch-related, inflammatory, and profibrotic responses were also accentuated in male mice, as shown by higher transcript levels of atrial natriuretic peptide, monocyte chemoattractant protein-1, and transforming growth factor-β. Our results support sex-specific regulation of the calcineurin pathway in response to largely blood pressure–independent mineralocorticoid action. We suggest that sex-specific calcineurin activation determines the maladaptive cardiac and renal hypertrophic responses and accompanying organ injury in male mice.

Mononuclear cells (MNCs) are the primary cell type involved in the pro-inflammatory state of obesity-linked insulin-resistance, and atherosclerosis. Increased serum levels of MMP-9 are reported in insulin-resistant type 2 diabetic patients. Here we demonstrate insulin facilitating human monocytic THP-1 cell chemotaxis via prolonged Erk1/2-dependent induction of MMP-9. In vivo, significantly increased serum levels of MMP-9 were found in obesity-induced hyperinsulinemic C57BL/J6 mice, which were diminished by treatment with the anti-diabetic PPARγ-ligand pioglitazone. In line with this, pioglitazone inhibited Erk1/2-phosphorylation and subsequent insulin-dependent MMP-9 synthesis in THP-1 cells. Thus, insulin increases MMP-9 gelatinolytic activity in monocytic cells, which results in accelerated chemotaxis. Hyperinsulinemia is associated with enhanced MMP-9 serum levels, potentially facilitating monocyte migration to and infiltration of adipose tissue and the arterial wall, thereby contributing to the increased cardiovascular risk in obese, hyperinsulinemic patients.

In weight loss trials, a considerable inter-individual variability in reduction of fat mass and changes of insulin resistance is observed, even under standardized study conditions. The underlying mechanisms are not well understood. Given the metabolic properties of the atrial natriuretic peptide (ANP) system, we hypothesized that ANP signaling might be involved in this phenomenon by changes of ANP secretion or receptor balance. Therefore, we investigated the impact of systemic, adipose and myocellular ANP system on metabolic and anthropometric improvements during weight loss.We comprehensively investigated 143 subjects (31 male, 112 female) before and after a 3 month-standardized weight loss program. The time course of BMI, fat mass, insulin sensitivity, circulating mid-regional proANP (MR-proANP) levels as well as adipose and myocellular natriuretic receptor A (NPR-A) and C (NPR-C) mRNA expression were investigated.BMI decreased by -12.6±3.7%. This was accompanied by a remarkable decrease of adipose NPR-C expression (1005.0±488.4 vs. 556.7±465.6; p0.001) as well as a tendency towards increased adipose NPR-A expression (4644.7±946.8 vs. 4877.6±869.8; p=0.051). Weight loss induced changes in NPR-C (ΔNPR-C) was linked to relative reduction of total fat mass (ΔFM) (r=0.281; p0.05), reduction of BMI (r=0.277; p0.01), and increase of free fatty acids (ΔFFA) (r=-0.258; p0.05). Basal NPR-C expression and weight loss induced ΔNPR-C independently explained 22.7% of ΔFM. In addition, ΔMR-proANP was independently associated with improvement of insulin sensitivity (standardized ß=0.246, p0.01).ANP receptor expression predicted the degree of weight loss induced fat mass reduction. Our comprehensive human data support that peripheral ANP signalling is involved in control of adipose tissue plasticity and function during weight loss. (Funded by the Deutsche Forschungsgemeinschaft (KFO281/2), the Berlin Institute of Health (BIH) and the German Centre for Cardiovascular Research (DZHK/BMBF); ClinicalTrials.gov number: NCT00850629).

Insulin resistance plays a crucial role in the development of type 2 diabetes. Insulin receptor signalling is antagonized and tightly controlled by protein tyrosine phosphatases (PTPs). However, the precise role of the PTP src homology 2 domain-containing phosphatase 1 (SHP-1) in insulin resistance has not been explored. Male C57BL/6J mice were fed a high-fat diet (HFD, 60% kcal from fat), to induce insulin resistance, or a low-fat diet (LFD, 10% kcal from fat) for 10 weeks. Afterwards, HFD-fed mice were pharmacologically treated with the SHP-1 (Ptpn6) inhibitor sodium stibogluconate and the broad spectrum pan-PTP inhibitor bis(maltolato)oxovanadium(IV) (BMOV). Both inhibitors ameliorated the metabolic phenotype, as evidenced by reduced body weight, improved insulin sensitivity and glucose tolerance, which was not due to altered PTP gene expression. In parallel, phosphorylation of the insulin receptor and of the insulin signalling key intermediate Akt was enhanced, and both PTP inhibitors and siRNA-mediated SHP-1 downregulation resulted in an increased glucose uptake in vitro. Finally, recombinant SHP-1 was capable of dephosphorylating the ligand-induced tyrosine-phosphorylated insulin receptor. These results indicate a central role of SHP-1 in insulin signalling during obesity, and SHP-1 inhibition as a potential therapeutic approach in metabolic diseases.

Endurance exercise training induces substantial adaptive cardiac modifications such as left ventricular hypertrophy (LVH). Simultaneously to the development of LVH, adipose tissue (AT) lipolysis becomes elevated upon endurance training to cope with enhanced energy demands. In this study, we investigated the impact of adipose tissue lipolysis on the development of exercise-induced cardiac hypertrophy. Mice deficient for adipose triglyceride lipase (Atgl) in AT (atATGL-KO) were challenged with chronic treadmill running. Exercise-induced AT lipolytic activity was significantly reduced in atATGL-KO mice accompanied by the absence of a plasma fatty acid (FA) increase. These processes were directly associated with a prominent attenuation of myocardial FA uptake in atATGL-KO and a significant reduction of the cardiac hypertrophic response to exercise. FA serum profiling revealed palmitoleic acid (C16:1n7) as a new molecular co-mediator of exercise-induced cardiac hypertrophy by inducing nonproliferative cardiomyocyte growth. In parallel, serum FA analysis and echocardiography were performed in 25 endurance athletes. In consonance, the serum C16:1n7 palmitoleate level exhibited a significantly positive correlation with diastolic interventricular septum thickness in those athletes. No correlation existed between linoleic acid (18:2n6) and diastolic interventricular septum thickness. Collectively, our data provide the first evidence that adipose tissue lipolysis directly promotes the development of exercise-induced cardiac hypertrophy involving the lipokine C16:1n7 palmitoleate as a molecular co-mediator. The identification of a lipokine involved in physiological cardiac growth may help to develop future lipid-based therapies for pathological LVH or heart failure.

The prevalence of obesity and obesity-related metabolic and cardiovascular disorders such as type 2 diabetes mellitus, dyslipidemia, and obesity-mediated hypertension is rising constantly. As the metabolic manifestation of those disorders differs in their etiology and kinetics, the pharmacological approaches to improve metabolic outcome of those diseases are very limited. The renin–angiotensin–­aldosterone system seems to be directly involved in pathophysiology of obesity and associated diseases. Those processes are mainly mediated by the increased activity of the angiotensin type 1 receptor (AT 1 R), which negatively regulates glucose and lipid metabolism, insulin sensitivity, and adipose tissue remodeling. Moreover, AT 1 R inhibitors (ARBs), used as a standard therapy to treat hypertension and related end-­organ damages, were demonstrated to possess some antidiabetogenic properties. The metabolic effects mediated by the second angiotensin type 2 receptor (AT 2 R) remain elusive, although the first experiments using direct AT 2 R agonists in vitro and in vivo indicate a beneficial role of this receptor in the regulation of glucose and lipid metabolism, mostly due to their potent anti-inflammatory characteristic.

Background: The peroxisome proliferator-activated receptor alpha (PPARα) is a central regulator of myocardial fatty acid (FA) metabolism implicated in the pathogenesis of heart failure. Aims: To characterize PPARα regulation in human dilated cardiomyopathy (DCM), we studied the expression of cardiac PPARα, cardiac carnitine palmitoyl-transferase I (CPT-1), a major PPARα target gene, and of the cardiac glucose transporter GLUT-4 in patients with DCM. Methods: Left ventricular biopsies were taken from patients with DCM (n=16) and control subjects (n=15), and mRNA expression was quantitated using real-time PCR (SYBR®Green) and protein expression was measured by Western immunoblotting. Results: Left ventricular PPARα mRNA levels were significantly increased in the DCM group compared to the control group (136±25.4% vs. control, p

Selective peroxisome proliferator–activated receptor (PPAR) γ modulation is a new pharmacological approach that, based on selective receptor-cofactor interactions and target gene regulation, should result in potent insulin sensitization in the absence of PPARγ-mediated adverse effects. Here, we characterize two angiotensin receptor blockers (ARBs), telmisartan and irbesartan, as new selective PPAR modulators (SPPARMs). Analysis of PPARγ protein conformation using protease protection showed that telmisartan directly interacts with the receptor, producing a distinct conformational change compared with a glitazone. Glutathione S-transferase pull-down and fluorescence resonance energy transfer assays revealed selective cofactor binding by the ARBs compared with glitazones with an attenuated release of the nuclear receptor corepressor and absence of transcriptional intermediary factor 2 recruitment by ARBs. Consistently, selective cofactor binding resulted in differential gene expression profiles in adipocytes (ARB versus glitazone treated) assessed by oligo microarray analysis. Finally, telmisartan improved insulin sensitivity in diet-induced obese mice in the absence of weight gain. The present study identifies two ARBs as new SPPARMs. SPPARM activity by ARBs could retain the metabolic efficacy of PPARγ activation with reduction in adverse effects exerting in parallel AT1 receptor blockade. This may provide a new therapeutic option for better cardiovascular risk management in metabolic diseases and may initiate the development of new classes of drugs combining potent antihypertensive and antidiabetic actions.

HMG-CoA reductase inhibitors have direct vascular effects that contribute to plaque stability. In the current study, the authors demonstrate that the HMG-CoA reductase inhibitors atorvastatin and pravastatin augment the adhesion of human (HSMCs) and rat aortic smooth muscle cells (RASMCs) to collagen I via induction of alpha2beta1-integrin receptors. Atorvastatin (0.1 microM ) increased the adhesion of HSMCs to collagen I up to 2-fold (p < 0.01) and pravastatin (1.0 microM ) up to 1.8-fold (p < 0.01) after treatment of at least 24 h. This increase in adhesion was concentration dependent and was observed for treatment periods from 16 to 72 h. Inhibition of isoprenoid synthesis with mevalonate and geranyl-geraniol prevented the statin-induced effect on human and rat smooth muscle cells. Flow cytometry revealed an increased expression of alpha2- and beta1-integrins after treatment with atorvastatin (0.1 microM ) at 24 and 48 h. Atorvastatin increased levels of beta1-integrin mRNA after 12- and 24-h treatment in HSMCs, which was inhibited by mevalonate. Furthermore, atorvastatin (0.1 microM ) and pravastatin (1.0 microM ) inhibited chemotaxis of HSMCs on collagen I, which was also reversed by mevalonate treatment. In contrast, inhibition of beta1-integrins with a specific antibody nearly doubled (p < 0.01) the rate of chemotaxis. These data indicate that the chemotactic activity in HSMCs is inhibited in part by up-regulation of alpha2beta1-integrin receptors. The current study indicates that HMG-CoA reductase inhibitors increase cell-matrix interaction with collagen I via induction of alpha2beta1-integrins and increased adhesion to collagen I.

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose metabolism and exert several vascular effects that may provide a dual benefit of these receptors on metabolic disorders and atherosclerotic vascular disease. Endothelial cell migration is a key event in the pathogenesis of atherosclerosis. We therefore investigated the effects of lipid-lowering PPARa-activators (fenofibrate, WY14643) and antidiabetic PPARc-activators (troglitazone, ciglitazone) on this endothelial cell function. Both PPARa- and PPARc-activators significantly inhibited VEGF-induced migration of human umbilical vein endothelial cells (EC) in a concentration-dependent manner. Chemotactic signaling in EC is known to require activation of two signaling pathways: the phosphatidylinositol-3-kinase ðPI3K Þ! Akt- and the ERK1/2 mitogen-activated protein kinase (ERK MAPK) pathway. Using the pharmacological PI3K-inhibitor wortmannin and the ERK MAPK-pathway inhibitor PD98059, we observed a complete inhibition of VEGF-induced EC migration. VEGF-induced Akt phosphorylation was significantly inhibited by both PPARa- and c-activators. In contrast, VEGF-stimulated ERK MAPK-activation was not affected by any of the PPAR-activators, indicating that they inhibit migration either downstream of ERK MAPK or independent from this pathway. These results provide first evidence for the antimigratory effects of PPAR-activators in EC. By inhibiting EC migration PPAR-activators may protect the vasculature from pathological alterations associated with metabolic disorders. 2002 Elsevier Science (USA). All rights reserved.

AIM To investigate matrix metalloproteinase-11 (MMP-11) expression in adipose tissue dysfunction, using in vitro and in vivo models of insulin resistance. METHODS Culture of mouse 3T3-L1 preadipocytes were induced to differentiation into mature 3T3-L1 adipocytes. Cellular insulin resistance was induced by treating differentiated cultured adipocytes with hypoxia and/or tumor necrosis factor (TNF)-α, and transcriptional changes were analyzed in each condition thereafter. For the in vivo studies, MMP-11 expression levels were measured in white adipose tissue (WAT) from C57BL/6J mice that underwent low fat diet or high-fat feeding in order to induce obesity and obesity-related insulin resistance. Statistical analysis was carried out with GraphPad Prism Software. RESULTS MMP-11 mRNA expression levels were significantly higher in insulin resistant 3T3-L1 adipocytes compared to control cells (1.46 ± 0.49 vs 0.83 ± 0.21, respectively; P < 0.00036). The increase in MMP-11 expression was observed even in the presence of TNF-α alone (3.79 ± 1.11 vs 1 ± 0.17, P < 0.01) or hypoxia alone (1.79 ± 0.7 vs 0.88 ± 0.1, P < 0.00023). The results obtained in in vitro experiments were confirmed in the in vivo model of insulin resistance. In particular, MMP-11 mRNA was upregulated in WAT from obese mice compared to lean mice (5.5 ± 2.8 vs 1.1 ± 0.7, respectively; P < 3.72E-08). The increase in MMP-11 levels in obese mice was accompanied by the increase in typical markers of fibrosis, such as collagen type VI alpha 3 (Col6α3), and fibroblast-specific protein 1. CONCLUSION Our results indicate that dysregulation of MMP-11 expression is an early process in the adipose tissue dysfunction, which leads to obesity and obesity-related insulin resistance.

Background: Insulin resistance plays a crucial role in the development of type 2 diabetes, and exerts great impact on vascular inflammation and remodeling. At the molecular level a post-insulin receptor (IR) defect in insulin signaling has been suggested to contribute to insulin resistance. IR signaling is antagonized and tightly controlled by protein tyrosine phosphatases (PTPs). The precise role of PTPs in insulin resistance, however, has not been explored. Results: Male C57BL/6J mice were fed a high-fat diet (HFD, 60% kcal from fat) to induce insulin resistance, or a low-fat diet (LFD, 10% kcal from fat) for 10 weeks. Afterwards, HFD mice were treated with PTP-inhibitors for additional 6 weeks. Mice under HFD exhibited a significant increase in body weight as well as decreased respiratory quotient and adiponectin levels, and were characterized by impaired insulin- and glucose tolerance. Organ-based gene expression analyses in insulin-resistant mice demonstrated upregulation of SHP-1, PTP1B, LAR, and DEP-1 in insulin-sensitive organs. SHP-1 was further explored in vitro. Insulin stimulation in murine liver cells induced site-selective hyper-phosphorylation at IR tyrosine-sites Y1158, and Y1361 after inhibition of SHP-1. Furthermore, SHP-1 impairment time-dependently enhanced insulin-induced Akt- and Erk-phosphorylation, and resulted in elevated glucose uptake in skeletal muscle cells. Administration of a SHP-1 inhibitor (Sodium Stibogluconate) and a brought pan-PTP inhibitor (BMOV) in HFD mice led to improvement of both insulin- and glucose tolerance. In accordance, PTP-activity was significantly impaired in epididymal fat, skeletal muscle, and liver under BMOV treatment, being confirmed by reduced ex vivo dephosphorylation of a radioactive labelled peptide (AEEEIYGEFEAKKKK). Finally, BMOV- and SHP-1 treatment also resulted in reduced body weight. Conclusions: IR-antagonizing PTPs were organ-specifically regulated in insulin resistance. The results indicate a central role of PTPs and, in particular, of SHP-1 as endogenous antagonists of the IR. Taken together targeting PTPs led to beneficial effects in insulin resistance, and may thus improve metabolic diseases as well as cardiovascular morbidity and mortality.

Introduction: Myocardial metabolism undergoes change in response to pathological cardiac hypertrophy (PH), characterized by increased reliance on glucose oxidation, decreased free fatty acid (FFA) oxidation and a loss of metabolic flexibility. Cardiac metabolism is influenced by other organs such as adipose tissue. Hence, we aimed to investigate the effect of Adipose Triglyceride Lipase (ATGL) in adipose tissue on the development of PH and heart failure (HF) in a pressure overload-induced cardiac hypertrophy model in mice. Methods: Male adipose tissue specific ATGL-knock out (atATGL-KO) and wild type mice (WT) underwent sham surgery (sham) or transverse aortic constriction (TAC). After 11 weeks, mice were sacrificed and organs were harvested. We performed echocardiography one week before and 11 weeks after surgery. Left ventricular mass (LVM), left ventricular mass/tibia length (LVM/TL) and ejection fraction (EF) were calculated. Beta-myosin heavy chain (β-MyHC) was measured in RNA of hearts. Insulin resistance was assessed by an intraperitoneal glucose tolerance test (GTT) and an insulin tolerance test (ITT). FFAs were measured in serum in total. Results: LVM and LVM/TL in WT was significantly higher compared to atATGL-KO after TAC (LVM/TL [mg/mm] WT-TAC: 18,0±2,2; atATGL-KO-TAC: 13,1±2,3; p Beta-MyHC, a marker for PH, was markedly higher in WT-TAC than in atATGL-KO-TAC (WT-TAC: 11,3±3,6; atATGL-KO-TAC: 1.9±0,6; p While WT-TAC showed higher Serum FFA-levels than atATGL-KO-TAC ([mmol/l] WT-TAC: 0,97±0,086; atATGL-KO-TAC: 0,49±0,032; p Conclusion: The present study demonstrates that atATGL is a crucial determinant for the development of pressure overload-induced PH and HI. The lack of ATGL in adipose tissue, the associated reduction of fatty acid release in the circulation and subsequent switches in cardiac energy substrates from free fatty acids to glucose are potential underlying mechanisms of this process.

The G protein-coupled receptor 30 (GPR30) has been claimed as an estrogen receptor. However, the literature reports controversial findings and the physiological function of GPR30 is not fully understood yet. Consistent with studies assigning a role of GPR30 in the cardiovascular and metabolic systems, GPR30 expression has been reported in small arterial vessels, pancreas and chief gastric cells of the stomach. Therefore, we hypothesized a role of GPR30 in the onset and progression of cardiovascular and metabolic diseases. In order to test our hypothesis, we investigated the effects of a high-fat diet on the metabolic and cardiovascular profiles of Gpr30-deficient mice (GPR30-lacZ mice). We found that GPR30-lacZ female, rather than male, mice had significant lower levels of HDL along with an increase in fat liver accumulation as compared to control mice. However, two indicators of cardiac performance assessed by echocardiography, ejection fraction and fractional shortening were both decreased in an age-dependent manner only in Gpr30-lacZ male mice. Collectively our results point to a potential role of Gpr30 in preserving lipid metabolism and cardiac function in a sex- and age-dependent fashion. (C) 2014 Elsevier B.V. All rights reserved.

Vascular smooth muscle cell (VSMC) migration involves adhesion, locomotion, and invasion regulated by various signaling molecules, among which the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) play a critical role. We have shown that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands troglitazone and rosiglitazone inhibit VSMC migration downstream of ERK MAPK. The purpose of the current study was to more specifically determine which step(s) in VSMC migration are targeted by inhibition of the ERK MAPK pathway or activation of PPAR-gamma. VSMC adhesion was not affected by the ERK MAPK pathway inhibitor PD98059 or PPAR-gamma ligands. Phosphorylation and activation of myosin light chain kinase (MLCK) play important roles in cell locomotion. Platelet-derived growth factor (PDGF)-induced MLCK phosphorylation (1.7-fold) was completely blocked by PD98059 at 30 microM (p < 0.05), but not by troglitazone or rosiglitazone. PDGF-directed migration (5.8-fold) was inhibited by PD98059 (-88% at 30 microM) and the MLCK inhibitor ML9 (0.1-1 microM, -84% at 1 microM) (all p < 0.05). The transcription factor Ets-1 mediates matrix metalloproteinase induction required for tissue invasion by VSMC. PDGF (20 ng/ml) stimulated an Ets-1 protein expression (14-fold at 60 min) in VSMC, which was inhibited by PD98059 (-72% at 30 microM), troglitazone (-69% at 20 microM), and rosiglitazone (-54% at 10 microM) (all p < 0.05). Immunohistochemistry of rat aortae 2 h after balloon injury showed a dramatic upregulation of Ets-1, which was markedly inhibited in animals that had received troglitazone treatment. In contrast, phosphorylated ERK MAPK was not affected by troglitazone. These data are consistent with PPAR-gamma ligands exerting their anti-migratory effects downstream of ERK MAPK activation by blocking nuclear events, such as Ets-1 expression, required for cell invasion in response to arterial injury.

Abstract —Retinoids inhibit rat vascular smooth muscle cell (VSMC) proliferation in vitro and intimal hyperplasia in vivo. We examined the mechanism of the antiproliferative effect of retinoids on human coronary artery smooth muscle cells (human CASMCs). The RAR ligands all- trans -retinoic acid (atRA) and ethyl- p -[( E )-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-l-propenyl]-benzoic acid (TTNPB); a pan-RXR/RAR agonist, 9- cis -retinoic acid (9cRA); and the RXR-selective ligand AGN4204 all inhibited DNA synthesis stimulated with platelet-derived growth factor and insulin (IC 50 : TTNPB 63 nmol/L, atRA 120 nmol/L, AGN4204 460 nmol/L, 9cRA 1.5 μmol/L). All retinoids blocked cell cycle progression as determined by flow cytometry and inhibited retinoblastoma protein (Rb) phosphorylation. TTNPB, atRA, and AGN4204 inhibited the mitogenic induction of cyclin D1, whereas 9cRA had no effect. None of the retinoids affected the expression of CDK 2, 4, or 6 or cyclin E. All retinoids attenuated mitogen-induced downregulation of CDKI p27 Kip1 , a major negative regulator of Rb phosphorylation, partly through stabilizing p27 Kip1 turnover. These data demonstrate that retinoids have antiproliferative activity by modulating G 1 → S cell cycle regulators in human CASMCs through inhibition of Rb phosphorylation and elevation of p27 Kip1 levels.

Abstract —Peroxisome proliferator–activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor expressed in all of the major cell types found in atherosclerotic lesions: monocytes/macrophages, endothelial cells, and smooth muscle cells. In vitro, PPARγ ligands inhibit cell proliferation and migration, 2 processes critical for vascular lesion formation. In contrast to these putative antiatherogenic activities, PPARγ has been shown in vitro to upregulate the CD36 scavenger receptor, which could promote foam cell formation. Thus, it is unclear what impact PPARγ activation will have on the development and progression of atherosclerosis. This issue is important because thiazolidinediones, which are ligands for PPARγ, have recently been approved for the treatment of type 2 diabetes, a state of accelerated atherosclerosis. We report herein that the PPARγ ligand, troglitazone, inhibited lesion formation in male low density lipoprotein receptor–deficient mice fed either a high-fat diet, which also induces type 2 diabetes, or a high-fructose diet. Troglitazone decreased the accumulation of macrophages in intimal xanthomas, consistent with our in vitro observation that troglitazone and another thiazolidinedione, rosiglitazone, inhibited monocyte chemoattractant protein-1–directed transendothelial migration of monocytes. Although troglitazone had some beneficial effects on metabolic risk factors (in particular, a reduction of insulin levels in the diabetic model), none of the systemic cardiovascular risk factors was consistently improved in either model. These observations suggest that the inhibition of early atherosclerotic lesion formation by troglitazone may result, at least in part, from direct effects of PPARγ activation in the artery wall.

Glitazones or thiazolidinediones (TZDs) are insulin-sensitizing drugs widely used in oral antidiabetic therapy ([1][1]). Currently, 2 substances, pioglitazone and rosiglitazone, are available in daily clinical practice. Both glitazones act as ligands for the nuclear hormone receptor peroxisome

Background Matrix metalloproteinase (MMP)-dependent extracellular matrix (ECM) remodeling is a key feature in cardiometabolic syndrome-associated adipogenesis and atherosclerosis. Activation of membrane-tethered (MT) 1-MMP depends on furin (PCSK3). However, the regulation and function of the natural furin-inhibitor serpinB8 and thus furin/MT1-MMP-activity in obesity-related tissue inflammation/remodeling is unknown. Here we aimed to determine the role of serpinB8/furin in obesity-associated chronic inflammation. Methods and Results Monocyte → macrophage transformation was characterized by decreases in serpinB8 and increases in furin/MT1-MMP. Rescue of serpinB8 by protein overexpression inhibited furin-dependent pro-MT1-MMP activation in macrophages, supporting its role as a furin-inhibitor. Obese white adipose tissue-facilitated macrophage migration was inhibited by furin- and MMP-inhibition, stressing the importance of the furin-MMP axis in fat tissue inflammation/remodeling. Monocytes from obese patients (body mass index (BMI) >30kg/m2) had higher furin, MT1-MMP, and resistin gene expression compared to normal weight individuals (BMI

Background Insulin signaling is tightly controlled by tyrosine dephosphorylation of the insulin receptor through protein-tyrosine-phosphatases (PTPs). DEP-1 is a PTP dephosphorylating tyrosine residues in a variety of receptor tyrosine kinases. Here, we analyzed whether DEP-1 activity is differentially regulated in liver, skeletal muscle and adipose tissue under high-fat diet (HFD), examined the role of DEP-1 in insulin resistance in vivo, and its function in insulin signaling. Results Mice were fed an HFD for 10 weeks to induce obesity-associated insulin resistance. Thereafter, HFD mice were subjected to systemic administration of specific antisense oligonucleotides (ASOs), highly accumulating in hepatic tissue, against DEP-1 or control ASOs. Targeting DEP-1 led to improvement of insulin sensitivity, reduced basal glucose level, and significant reduction of body weight. This was accompanied by lower insulin and leptin serum levels. Suppression of DEP-1 in vivo also induced hyperphosphorylation in the insulin signaling cascade of the liver. Moreover, DEP-1 physically associated with the insulin receptor in situ, and recombinant DEP-1 dephosphorylated the insulin receptor in vitro. Conclusions These results indicate that DEP-1 acts as an endogenous antagonist of the insulin receptor, and downregulation of DEP-1 results in an improvement of insulin sensitivity. DEP-1 may therefore represent a novel target for attenuation of metabolic diseases.