Verena Benz

Histone Deacetylase 6 (HDAC6) Is an Essential Modifier of Glucocorticoid-Induced Hepatic Gluconeogenesis

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.

Sex differences in physiological cardiac hypertrophy are associated with exercise-mediated changes in energy substrate availability

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.

Sexual Dimorphic Regulation of Body Weight Dynamics and Adipose Tissue Lipolysis

Background Successful reduction of body weight (BW) is often followed by recidivism to obesity. BW-changes including BW-loss and -regain is associated with marked alterations in energy expenditure (EE) and adipose tissue (AT) metabolism. Since these processes are sex-specifically controlled, we investigated sexual dimorphisms in metabolic processes during BW-dynamics (gain-loss-regain). Research Design Obesity was induced in C57BL/6J male (m) and female (f) mice by 15 weeks high-fat diet (HFD) feeding. Subsequently BW was reduced (-20%) by caloric restriction (CR) followed by adaptive feeding, and a regain-phase. Measurement of EE, body composition, blood/organ sampling were performed after each feeding period. Lipolysis was analyzed ex-vivo in gonadal AT. Results Male mice exhibited accelerated BW-gain compared to females (relative BW-gain m:140.5±3.2%; f:103.7±6.5%; p<0.001). In consonance, lean mass-specific EE was significantly higher in females compared to males during BW-gain. Under CR female mice reached their target-BW significantly faster than male mice (m:12.2 days; f:7.6 days; p<0.001) accompanied by a sustained sex-difference in EE. In addition, female mice predominantly downsized gonadal AT whereas the relation between gonadal and total body fat was not altered in males. Accordingly, only females exhibited an increased rate of forskolin-stimulated lipolysis in AT associated with significantly higher glycerol concentrations, lower RER-values, and increased AT expression of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Analysis of AT lipolysis in estrogen receptor alpha (ERα)–deficient mice revealed a reduced lipolytic rate in the absence of ERα exclusively in females. Finally, re-feeding caused BW-regain faster in males than in females. Conclusion The present study shows sex-specific dynamics during BW-gain-loss-regain. Female mice responded to CR with an increase in lipolytic activity, and augmented lipid-oxidation leading to more efficient weight loss. These processes likely involve ERα-dependent signaling in AT and sexual dimorphic regulation of genes involved in lipid metabolism.

Sexual dimorphism in obesity-mediated left ventricular hypertrophy

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.

Sex-Specific Differences in Type 2 Diabetes Mellitus and Dyslipidemia Therapy: PPAR Agonists

The influence of sex on the development of obesity, Type 2 Diabetes Mellitus (T2DM), and dyslipidemia is well documented, although the molecular mechanism underlying those differences reminds elusive. Ligands of peroxisome proliferator-activated receptors (PPARs) are used as oral antidiabetics (PPARgamma agonists: thiazolidinediones, TZDs), or for the treatment of dyslipidemia and cardiovascular diseases, due to their lipid-lowering properties (PPARalpha agonists: fibrates), as PPARs control transcription of a set of genes involved in the regulation of lipid and carbohydrate metabolism. Given a high prevalence of those metabolic disorders, and thus a broad use of PPAR agonists, the present review will discuss distinct aspects of sex-specific differences in antiobesity treatment using those groups of PPAR ligands.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin–angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1–7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that—at least in astrocytes—both receptors functionally depend on each other.

Adipose Tissue Lipolysis Promotes Exercise-induced Cardiac Hypertrophy Involving the Lipokine C16:1n7-Palmitoleate

Background: Endurance training induces physiological cardiac hypertrophy and elevates adipose tissue lipolysis. Results: Adipose-specific adipose triglyceride lipase (Atgl)-knock-out mice exhibit attenuated exercise-induced cardiac hypertrophy likely mediated by the lack of C16:1n7 palmitoleate actions on the heart. Conclusion: Atgl-mediated adipose lipolysis regulates physiological cardiac hypertrophy. Significance: Adipose-derived lipokines may serve as important molecular mediators of cardiac physiology and pathology. 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.

398 EVIDENCE OF A DIRECT MAS-AT2 RECEPTOR DIMERIZATION

Objectives and Background: The seven transmembrane G-protein coupled receptors MAS and AT2 (angiotensin type 2) seem to have a parallel, protective role in the renin angiotensin system suggesting functional interaction, but a molecular association between these two receptors has not been explored. Design and Methods: In the present study, the interaction between MAS and AT2 receptors was assessed by studies performed in living cells (HEK 293) using fluorescence resonance energy transfer (FRET).FRET pairs of MAS and AT2 fused in the C-terminus with CFP or YFP were designed. FRET efficiencies were determined by monitoring the increase in the CFP (FRET-donor) fluorescence emission during selective YFP (FRET-acceptor) photobleaching. Results: Our results show a significant FRET efficiency of 10.8 ± 0.8% when AT2-YFP and MAS-CFP (and vice versa) were coexpressed indicating a clear hetero-interaction/dimerization between the two receptors. Homodimer interactions were also explored and we observed that both MAS and AT2 receptors self-oligomerize, as demonstrated by FRET efficiencies of 7.4 ± 0.8% and 9.2 ± 0.8%, respectively. The interactions of the receptors are specific as (i) no FRET efficiency was observed with an unrelated but colocalized transmembrane receptor and (ii) expression of non-fluorescent MAS and AT2 receptors competed with FRET efficiencies. Conclusions: The evidence provided in this study suggests that MAS and AT2 receptors form oligomerized complexes in the membrane, a property that could influence the functionality of the receptors and their substrate selectivity.

High-Mobility Group A1 Protein

Rationale: The nuclear receptor peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) is an important regulator of gene transcription in vascular cells and mediates the vascular protection observed with antidiabetic glitazones. Objective: To determine the molecular mechanism of ligand-dependent transrepression in vascular smooth muscle cells and their impact on the vascular protective actions of PPAR&ggr;. Methods and Results: Here, we report a molecular pathway in vascular smooth muscle cells by which ligand-activated PPAR&ggr; represses transcriptional activation of the matrix-degrading matrix metalloproteinase-9 (MMP-9) gene, a crucial mediator of vascular injury. PPAR&ggr;-mediated transrepression of the MMP-9 gene was dependent on the presence of the high-mobility group A1 (HMGA1) protein, a gene highly expressed in vascular smooth muscle cells, newly identified by oligonucleotide array expression analysis. Transrepression of MMP-9 by PPAR&ggr; and regulation by HMGA1 required PPAR&ggr; SUMOylation at K367. This process was associated with formation of a complex between PPAR&ggr;, HMGA1, and the SUMO E2 ligase Ubc9 (ubiquitin-like protein SUMO-1 conjugating enzyme). After PPAR&ggr; ligand stimulation, HMGA1 and PPAR&ggr; were recruited to the MMP-9 promoter, which facilitated binding of SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), a nuclear corepressor involved in transrepression. The relevance of HMGA1 for vascular PPAR&ggr; signaling was underlined by the complete absence of vascular protection through a PPAR&ggr; ligand in HMGA1−/− mice after arterial wire injury. Conclusions: The present data suggest that ligand-dependent formation of HMGA1-Ubc9-PPAR&ggr; complexes facilitates PPAR&ggr; SUMOylation, which results in the prevention of SMRT corepressor clearance and induction of MMP-9 transrepression. These data provide new information on PPAR&ggr;-dependent vascular transcriptional regulation and help us to understand the molecular consequences of therapeutic interventions with PPAR&ggr; ligands in the vasculature.Rationale: The nuclear receptor peroxisome proliferator–activated receptor-γ (PPARγ) is an important regulator of gene transcription in vascular cells and mediates the vascular protection observed with antidiabetic glitazones. Objective: To determine the molecular mechanism of ligand-dependent transrepression in vascular smooth muscle cells and their impact on the vascular protective actions of PPARγ. Methods and Results: Here, we report a molecular pathway in vascular smooth muscle cells by which ligand-activated PPARγ represses transcriptional activation of the matrix-degrading matrix metalloproteinase-9 (MMP-9) gene, a crucial mediator of vascular injury. PPARγ-mediated transrepression of the MMP-9 gene was dependent on the presence of the high-mobility group A1 (HMGA1) protein, a gene highly expressed in vascular smooth muscle cells, newly identified by oligonucleotide array expression analysis. Transrepression of MMP-9 by PPARγ and regulation by HMGA1 required PPARγ SUMOylation at K367. This process was associated with formation of a complex between PPARγ, HMGA1, and the SUMO E2 ligase Ubc9 (ubiquitin-like protein SUMO-1 conjugating enzyme). After PPARγ ligand stimulation, HMGA1 and PPARγ were recruited to the MMP-9 promoter, which facilitated binding of SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), a nuclear corepressor involved in transrepression. The relevance of HMGA1 for vascular PPARγ signaling was underlined by the complete absence of vascular protection through a PPARγ ligand in HMGA1−/− mice after arterial wire injury. Conclusions: The present data suggest that ligand-dependent formation of HMGA1-Ubc9-PPARγ complexes facilitates PPARγ SUMOylation, which results in the prevention of SMRT corepressor clearance and induction of MMP-9 transrepression. These data provide new information on PPARγ-dependent vascular transcriptional regulation and help us to understand the molecular consequences of therapeutic interventions with PPARγ ligands in the vasculature. # Novelty and Significance {#article-title-36}

High-Mobility Group A1 Protein A New Coregulator of Peroxisome Proliferator–Activated Receptor-γ–Mediated Transrepression in the Vasculature

Rationale: The nuclear receptor peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) is an important regulator of gene transcription in vascular cells and mediates the vascular protection observed with antidiabetic glitazones. Objective: To determine the molecular mechanism of ligand-dependent transrepression in vascular smooth muscle cells and their impact on the vascular protective actions of PPAR&ggr;. Methods and Results: Here, we report a molecular pathway in vascular smooth muscle cells by which ligand-activated PPAR&ggr; represses transcriptional activation of the matrix-degrading matrix metalloproteinase-9 (MMP-9) gene, a crucial mediator of vascular injury. PPAR&ggr;-mediated transrepression of the MMP-9 gene was dependent on the presence of the high-mobility group A1 (HMGA1) protein, a gene highly expressed in vascular smooth muscle cells, newly identified by oligonucleotide array expression analysis. Transrepression of MMP-9 by PPAR&ggr; and regulation by HMGA1 required PPAR&ggr; SUMOylation at K367. This process was associated with formation of a complex between PPAR&ggr;, HMGA1, and the SUMO E2 ligase Ubc9 (ubiquitin-like protein SUMO-1 conjugating enzyme). After PPAR&ggr; ligand stimulation, HMGA1 and PPAR&ggr; were recruited to the MMP-9 promoter, which facilitated binding of SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), a nuclear corepressor involved in transrepression. The relevance of HMGA1 for vascular PPAR&ggr; signaling was underlined by the complete absence of vascular protection through a PPAR&ggr; ligand in HMGA1−/− mice after arterial wire injury. Conclusions: The present data suggest that ligand-dependent formation of HMGA1-Ubc9-PPAR&ggr; complexes facilitates PPAR&ggr; SUMOylation, which results in the prevention of SMRT corepressor clearance and induction of MMP-9 transrepression. These data provide new information on PPAR&ggr;-dependent vascular transcriptional regulation and help us to understand the molecular consequences of therapeutic interventions with PPAR&ggr; ligands in the vasculature.Rationale: The nuclear receptor peroxisome proliferator–activated receptor-γ (PPARγ) is an important regulator of gene transcription in vascular cells and mediates the vascular protection observed with antidiabetic glitazones. Objective: To determine the molecular mechanism of ligand-dependent transrepression in vascular smooth muscle cells and their impact on the vascular protective actions of PPARγ. Methods and Results: Here, we report a molecular pathway in vascular smooth muscle cells by which ligand-activated PPARγ represses transcriptional activation of the matrix-degrading matrix metalloproteinase-9 (MMP-9) gene, a crucial mediator of vascular injury. PPARγ-mediated transrepression of the MMP-9 gene was dependent on the presence of the high-mobility group A1 (HMGA1) protein, a gene highly expressed in vascular smooth muscle cells, newly identified by oligonucleotide array expression analysis. Transrepression of MMP-9 by PPARγ and regulation by HMGA1 required PPARγ SUMOylation at K367. This process was associated with formation of a complex between PPARγ, HMGA1, and the SUMO E2 ligase Ubc9 (ubiquitin-like protein SUMO-1 conjugating enzyme). After PPARγ ligand stimulation, HMGA1 and PPARγ were recruited to the MMP-9 promoter, which facilitated binding of SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), a nuclear corepressor involved in transrepression. The relevance of HMGA1 for vascular PPARγ signaling was underlined by the complete absence of vascular protection through a PPARγ ligand in HMGA1−/− mice after arterial wire injury. Conclusions: The present data suggest that ligand-dependent formation of HMGA1-Ubc9-PPARγ complexes facilitates PPARγ SUMOylation, which results in the prevention of SMRT corepressor clearance and induction of MMP-9 transrepression. These data provide new information on PPARγ-dependent vascular transcriptional regulation and help us to understand the molecular consequences of therapeutic interventions with PPARγ ligands in the vasculature. # Novelty and Significance {#article-title-36}