Brown adipose tissue dysfunction promotes heart failure via a trimethylamine N-oxide-dependent mechanism.

Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.We thank Kaori Yoshida, Keiko Uchiyama, Satomi Kawai, Naomi Hatanaka, Yoko Sawaguchi, Runa Washio, Takako Ichihashi, Nanako Koike, Keiko Uchiyama, Masaaki Nameta (Niigata University), Kaori Igarashi, Kaori Saitoh, Keiko Endo, Hiroko Maki, Ayano Ueno, Maki Ohishi, Sanae Yamanaka, Noriko Kagata (Keio University) for their excellent technical assistance, C. Ronald Kahn (Joslin Diabetes Center and Harvard Medical School) for providing the BAT cell line, Evan Rosen (Harvard Medical School) for providing us Ucp-Cre mice, Kosuke Morikawa (Kyoto University), Tomitake Tsukihara (University of Hyogo) and Shinya Yoshikawa (University of Hyogo) for their professional opinions and suggestions. Tis work was supported by a Grant-in-Aid for Scientifc Research (A) (20H00533) from MEXT, AMED under Grant Numbers JP20ek0210114, and AMED-CREST under Grant Number JP20gm1110012, and Moonshot Research and Development Program (21zf0127003s0201), MEXT Supported Program for the Strategic Research Foundation at Private Universities Japan, Private University Research Branding Project, and Leading Initiative for Excellent Young Researchers, and grants from the Takeda Medical Research Foundation, the Vehicle Racing Commemorative Foundation, Ono Medical Research Foundation, and the Suzuken Memorial Foundation (to T.M.). Support was also provided by a Grants-in-Aid for Young Scientists (Start-up) (26893080), and grants from the Uehara Memorial Foundation, Kowa Life Science Foundation, Manpei Suzuki Diabetes Foundation, SENSHIN Medical Research Foundation, ONO Medical Research Foundation, Tsukada Grant for Niigata University Medical Research, Te Nakajima Foundation, SUZUKEN memorial foundation, HOKUTO Corporation, Mochida Memorial Foundation for Medical & Pharmaceutical Research, Grants-in-Aid for Encouragement of Young Scientists (A) (16H06244), Daiichi Sankyo Foundation of Life Science, AMED Project for Elucidating and Controlling Mechanisms of Aging and Longevity under Grant Number JP17gm5010002, JP18gm5010002, JP19gm5010002, JP20gm5010002, JP21gm5010002, Astellas Foundation for Research on Metabolic Disorders, Research grant from Naito Foundation, Te Japan Geriatrics Society (to I.S.); by a Grant-in-Aid for Scientifc Research (C) (19K08974), Yujin Memorial Grant, Sakakibara Memorial Research Grant from Te Japan Research Promotion Society for Cardiovascular Diseases, TERUMO Life Science Foundation, Kanae Foundation (to Y.Y.), JST ERATO (JPMJER1902), AMED-CREST (JP20gm1010009), the Takeda Science Foundation, the Food Science Institute Foundation (to S.F.), and by a grant from Bourbon (to T.M., I.S. and Y.Y.).S

Vascular Senescence in Cardiovascular and Metabolic Diseases

In mammals, aging is associated with accumulation of senescent cells. Stresses such as telomere shortening and reactive oxygen species induce “cellular senescence”, which is characterized by growth arrest and alteration of the gene expression profile. Chronological aging is associated with development of age-related diseases, including heart failure, diabetes, and atherosclerotic disease, and studies have shown that accumulation of senescent cells has a causative role in the pathology of these age-related disorders. Endothelial cell senescence has been reported to develop in heart failure and promotes pathologic changes in the failing heart. Senescent endothelial cells and vascular smooth muscle cells are found in atherosclerotic plaque, and studies indicate that these cells are involved in progression of plaque. Diabetes is also linked to accumulation of senescent vascular endothelial cells, while endothelial cell senescence per se induces systemic glucose intolerance by inhibiting skeletal muscle metabolism. A close connection between derangement of systemic metabolism and cellular senescence is also well recognized. Aging is a complex phenomenon, and there is no simple approach to understanding the whole process. However, there is accumulating evidence that cellular senescence has a central role in the development and progression of various undesirable aspects of aging. Suppression of cellular senescence or elimination of senescent cells reverses phenotypic changes of aging in several models, and proof-of-concept has been established that inhibiting accumulation of senescent cells could become a next generation therapy for age-related disorders. It is clear that cellular senescence drives various pathological changes associated with aging. Accordingly, further investigation into the role of this biological process in age-related disorders and discovery of senolytic compounds are important fields for future exploration

Boysenberry polyphenol inhibits endothelial dysfunction and improves vascular health.

Endothelial cells have an important role in maintaining vascular homeostasis. Age-related disorders (including obesity, diabetes, and hypertension) or aging per se induce endothelial dysfunction that predisposes to the development of atherosclerosis. Polyphenols have been reported to suppress age-related endothelial cell disorders, but their role in vascular function is yet to be determined. We investigated the influence of boysenberry polyphenol on vascular health under metabolic stress in a murine model of dietary obesity. We found that administration of boysenberry polyphenol suppressed production of reactive oxygen species (ROS) and increased production of nitric oxide (NO) in the aorta. It has been reported that p53 induces cellular senescence and has a crucial role in age-related disorders, including heart failure and diabetes. Administration of boysenberry polyphenol significantly reduced the endothelial p53 level in the aorta and ameliorated endothelial cell dysfunction in iliac arteries under metabolic stress. Boysenberry polyphenol also reduced ROS and p53 levels in cultured human umbilical vein endothelial cells (HUVECs), while increasing NO production. Uncoupled endothelial nitric oxide synthase (eNOS monomer) is known to promote ROS production. We found that boysenberry polyphenol reduced eNOS monomer levels both in vivo and in vitro, along with an increase of eNOS dimerization. To investigate the components of boysenberry polyphenol mediating these favorable biological effects, we extracted the anthocyanin fractions. We found that anthocyanins contributed to suppression of ROS and p53, in association with increased NO production and eNOS dimerization. In an ex vivo study, anthocyanins promoted relaxation of iliac arteries from mice with dietary obesity. These findings indicate that boysenberry polyphenol and anthocyanins, a major component of this polyphenol, inhibit endothelial dysfunction and contribute to maintenance of vascular homeostasis

Role of smooth muscle cell p53 in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by remodeling and narrowing of the pulmonary arteries, which lead to elevation of right ventricular pressure, heart failure, and death. Proliferation of pulmonary artery smooth muscle cells (PASMCs) is thought to be central to the pathogenesis of PAH, although the underlying mechanisms are still being explored. The protein p53 is involved in cell cycle coordination, DNA repair, apoptosis, and cellular senescence, but its role in pulmonary hypertension (PH) is not fully known. We developed a mouse model of hypoxia-induced pulmonary hypertension (PH) and found significant reduction of p53 expression in the lungs. Our in vitro experiments with metabolomic analyses and the Seahorse XF extracellular flux analyzer indicated that suppression of p53 expression in PASMCs led to upregulation of glycolysis and downregulation of mitochondrial respiration, suggesting a proliferative phenotype resembling that of cancer cells. It was previously shown that systemic genetic depletion of p53 in a murine PH model led to more severe lung manifestations. Lack of information about the role of cell-specific p53 signaling promoted us to investigate it in our mouse PH model with the inducible Cre-loxP system. We generated a mouse model with SMC-specific gain or loss of p53 function by crossing Myh11-Cre/ERT2 mice with floxed Mdm4 mice or floxed Trp53 mice. After these animals were exposed to hypoxia for 4 weeks, we conducted hemodynamic and echocardiographic studies. Surprisingly, the severity of PH was similar in both groups of mice and there were no differences between the genotypes. Our findings in these mice indicate that activation or suppression of p53 signaling in SMCs has a minor role in the pathogenesis of PH and suggest that p53 signaling in other cells (endothelial cells, immune cells, or fibroblasts) may be involved in the progression of this condition

Inhibition of dipeptidyl peptidase-4 ameliorates cardiac ischemia and systolic dysfunction by up-regulating the FGF-2/EGR-1 pathway

<div><p>Dipeptidyl peptidase 4 inhibitors are used worldwide in the management of diabetes, but their role in the prevention or treatment of cardiovascular disorders has yet to be defined. We found that linagliptin, a DPP-4 inhibitor, suppressed capillary rarefaction in the hearts of mice with dietary obesity. Metabolomic analysis performed with capillary electrophoresis/mass spectrometry (LC-MS/MS) showed that linagliptin promoted favorable metabolic remodeling in cardiac tissue, which was characterized by high levels of citrulline and creatine. DNA microarray analysis revealed that the cardiac tissue level of early growth response protein 1 (EGR-1), which activates angiogenesis, was significantly reduced in untreated mice with dietary obesity, while this decrease was inhibited by administration of linagliptin. Mature fibroblast growth factor 2 (FGF-2) has a putative truncation site for DPP-4 at the NH2-terminal, and LC-MS/MS showed that recombinant DPP-4 protein cleaved the NH2-terminal dipeptides of mature FGF-2. Incubation of cultured neonatal rat cardiomyocytes with FGF-2 increased Egr1 expression, while it was suppressed by recombinant DPP-4 protein. Furthermore, vascular endothelial growth factor-A had a critical role in mediating FGF-2/EGR-1 signaling. In conclusion, pharmacological inhibition of DPP-4 suppressed capillary rarefaction and contributed to favorable remodeling of cardiac metabolism in mice with dietary obesity.</p></div

Gamma-Aminobutyric Acid Signaling in Brown Adipose Tissue Promotes Systemic Metabolic Derangement in Obesity

Summary: Brown adipose tissue (BAT) is a metabolically active organ that contributes to the maintenance of systemic metabolism. The sympathetic nervous system plays important roles in the homeostasis of BAT and promotes its browning and activation. However, the role of other neurotransmitters in BAT homeostasis remains largely unknown. Our metabolomic analyses reveal that gamma-aminobutyric acid (GABA) levels are increased in the interscapular BAT of mice with dietary obesity. We also found a significant increase in GABA-type B receptor subunit 1 (GABA-BR1) in the cell membranes of brown adipocytes of dietary obese mice. When administered to obese mice, GABA induces BAT dysfunction together with systemic metabolic disorder. Conversely, the genetic inactivation or inhibition of GABA-BR1 leads to the re-browning of BAT under conditions of metabolic stress and ameliorated systemic glucose intolerance. These results indicate that the constitutive activation of GABA/GABA-BR1 signaling in obesity promotes BAT dysfunction and systemic metabolic derangement. : Brown adipose tissue (BAT) is a metabolically active organ important for systemic metabolism. Here, Ikegami et al. identify a role for gamma-aminobutyric acid (GABA) in metabolic and BAT dysfunction in obese mice and demonstrate that inhibition of GABA/GABA-BR1-mediated signaling and of mitochondrial calcium overload can restore BAT function in obesity. Keywords: brown adipose tissue, BAT, metabolome, gamma-aminobutyric acid, GABA, obesit

Characterization of the cardiac urea cycle and creatine pathway.

<p>(A) Metabolomic data on pathway enrichment analysis in cardiac tissue. KEGG categories enriched by 1.5-fold in mice on a high fat diet (HFD) receiving linagliptin, a DPP-4 inhibitor (HFD+DPP-4i) (n = 5), compared to untreated HFD mice are described (n = 5). (B, E, F) Metabolites of the urea cycle (B, E) or creatine pathway (F) were assessed in cardiac tissue by metabolomic analysis with capillary electrophoresis/ mass spectrometry (CE/MS) in mice fed normal chow (NC), HFD, or HFD+DPP-4i (n = 5,5,5). (C, D) Quantitative PCR study for <i>Nos3</i> (C)(n = 6,6,7) and nitric oxide level (D)(n = 8,8,5) in the cardiac tissues of indicated mice groups (n = 6,6,7). (G) Quantitative PCR study for enzymes related to urea cycle (<i>Ass1</i>, <i>Asl</i>, <i>Arg1</i>, <i>Arg2</i> and <i>Otc</i>) or creatine pathway (<i>Gatm</i> and <i>Gamt</i>) (n = 11,11,13). The description “Low” indicates CT value to be mostly over 35 or undetected. (H) Scheme summarizing the findings of metabolomic analysis and quantitative PCR. Metabolites or transcripts are displayed by using the following colors: red (significantly increased in HFD mice receiving linagliptin compared to untreated HFD mice), orange (increased in HFD mice receiving linagliptin, but not significantly), black (no change), and gray (not detected or low). Data were analyzed by 2-tailed Student’s <i>t</i>-test (A,G), with 2-way ANOVA followed by Tukey’s multiple comparison (B–F) except for arginosuccinate that was analyzed by Student’s <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01. All values represent the mean ± s.e.m. NS = not significant.</p

DPP-4 inhibits angiogenesis by suppressing FGF-2/EGR-1/VEGF-A signaling.

<p>(A) Known peptide sequences of the NH2-terminal of mature FGF-2 in mice, rats, and humans (upper panel). Lower panel indicates peptide sequences of the NH2-terminal of mature FGF-2 after incubation with DPP-4 (analyzed by LC-MS/MS). (B, C) Quantitative PCR for <i>Egr1</i> and <i>Vegfa</i> in neonatal rat ventricular myocytes (NRVMs) incubated with PBS, recombinant FGF-2 (FGF-2)(50 ng/ml, 2 hr), or DPP-4 (0.1 μg/ml, 2 hr) in Fig 4B (n = 4,4,4), and with FGF-2 (50 ng/ml, 8 hr) or DPP-4 (0.1 μg/ml, 8 hr) in Fig 4C(n = 4,4,4)). As for experiments in Fig 4B and C, FGF-2 was pre-incubated with DPP-4 in tube for totally 8 hours at 37°C before administration to cells in the FGF-2+DPP-4 group. (D) Tube formation by human umbilical vein endothelial cells (HUVECs) incubated with PBS or recombinant FGF-2 (FGF-2) (35 ng/ml for 12 hr), either with DPP-4 (0.1 μg/ml for 12 hr) or without DPP-4 (Con). Right panel indicates quantification of tube length for experiments shown in the left panel (n = 3,3,3). (E) Quantitative PCR for <i>Egr1</i> and <i>Vegfa</i> expression in neonatal rat ventricular myocytes (NRVMs) after introduction of control si-RNA (si-Con) or si-Egr1 (n = 4,4). (F) Scheme showing a summary of the present findings. Metabolic stress increases circulating DPP-4, which suppresses FGF-2/EGR-1/VEGF-A signaling in cardiac tissue, leading to capillary rarefaction and cardiac dysfunction. In addition to blocking DPP-4, linagliptin, a DPP-4 inhibitor (DPP-4i), increased citrulline and creatine levels in cardiac tissue, which may also have contributed to suppressing pathologic changes in the obese failing heart. Whether there is a link between altered metabolic profile and angiogenic response remains to be determined. Data were analyzed by the 2-tailed Student’s t-test (E), or 2-way ANOVA followed by Tukey’s multiple comparison test (B, C, D). *P<0.05, **P<0.01. All values represent the mean ± s.e.m. NS = not significant.</p