Sirt3KO Mice Research Articles

Setmelanotide Administration Improves Cardiac Systolic Function in Sirtuin-3 Deficient Mice

We previously showed that activation of the brain leptin-melanocortin system, particularly melanocortin-4 receptors (MC4R), markedly improves cardiac function in experimental models of heart failure (HF) via mechanisms that are still poorly understood. Sirtuin 3 (Sirt3) is an important regulator of mitochondrial oxidative phosphorylation and deficiency of Sirt3 has been implicated in cardiovascular diseases. We recently found increased Sirt3 expression as well as increased levels of NAD+, the main Sirt3 activator, in cardiomyocytes of rats treated with chronic intracerebroventricular infusion of leptin at a dose that does not spill over into the systemic circulation. Therefore, we hypothesized that stimulation of Sirt3 activity is required for the improvement of cardiac function during activation of the brain leptin-MC4R axis. To test this hypothesis, we first examined if chronic MC4R activation, using an MC4R agonist, setmelanotide, improves cardiac function in naïve Sirt3-deficient (Sirt3-KO) mice. Baseline cardiac function was assessed by echocardiography (VisualSonics-VEVO3100®) in male and female Sirt3-KO and wild-type (WT) control mice (n=5/group) at 20 weeks of age, and then weekly during setmelanotide treatment (2 mg/kg/day — i.p. via osmotic minipump model 1002) for 3 weeks. Chronic MC4R activation with setmelanotide transiently reduced food intake in both groups and promoted weight loss in Sirt3-KO mice. Setmelanotide treatment improved systolic function in WT mice as evidenced by increases in ejection fraction and fractional shortening (EF: 52.8±4.4 vs. 72.4±4.4%; FS: 26.9±2.7 vs. 42.8±6.4%, for baseline vs. week 3, respectively) and other measures of systolic function including circumferential and longitudinal strains. Despite showing a tendency for reduced systolic function compared to WT controls at baseline, Sirt3-KO mice also exhibited improvement in systolic function with setmelanotide administration (EF: 44.1±1.8 vs. 65.3±2.9% and FS: 21.4±1.0 vs. 35.1±2.1%). No major changes were observed in measures of diastolic function. These results suggest that in a non-HF model, MC4R activation does not require stimulation of Sirt3 to improve indices of systolic function. (1R01HL163076, 1R01DK121411, P20GM104357, P30GM149404, U54GM115428, and AHA-835218). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sirtuin 3 controls cardiac energetics and protects against oxidative stress in electromagnetic radiation-induced cardiomyopathy

Electromagnetic radiation can cause injuries to both the structures and functions of the heart. No therapy is currently available to inhibit these untoward effects. Mitochondrial energetic damage and oxidative stress are drivers of electromagnetic radiation-induced cardiomyopathy (eRIC); however, the pathways that mediate these events are poorly defined. Sirtuin 3 (SIRT3) has been emerged as a key target for maintaining mitochondrial redox potential and metabolism, but its role in eRIC remains unknown. Here, Sirt3-KO mice and cardiac-specific SIRT3 transgenic mice were subjected to the investigation of eRIC. We found that Sirt3 protein expression level was down-regulated in eRIC mice model. Sirt3-KO markedly exaggerated decreases in cardiac energetics and increases in oxidative stress in microwave irradiation (MWI)-stressed mice. Conversely, cardiac-specific SIRT3 overexpression protected the hearts from these effects and rescued cardiac malfunction. Mechanistically, Sirt3 maintained AMP-activated protein kinase (AMPK) signaling pathway in MWI-stressed hearts in vivo. In conclusion, electromagnetic radiation repressed SIRT3 expression and disturbed cardiac energetics and redox homeostasis. The increased SIRT3 expression and AMPK activation in vivo prevented eRIC, indicating that SIRT3 will be a potential therapeutic target for curative interventions in eRIC.

SIRT3 Deficiency Enhances Ferroptosis and Promotes Cardiac Fibrosis via p53 Acetylation.

Cardiac fibrosis plays an essential role in the development of diastolic dysfunction and contributes to heart failure with preserved ejection fraction (HFpEF). Our previous studies suggested Sirtuin 3 (SIRT3) as a potential target for cardiac fibrosis and heart failure. In the present study, we explored the role of SIRT3 in cardiac ferroptosis and its contribution to cardiac fibrosis. Our data showed that knockout of SIRT3 resulted in a significant increase in ferroptosis, with increased levels of 4-hydroxynonenal (4-HNE) and downregulation of glutathione peroxidase 4 (GPX-4) in the mouse hearts. Overexpression of SIRT3 significantly blunted ferroptosis in response to erastin, a known ferroptosis inducer, in H9c2 myofibroblasts. Knockout of SIRT3 resulted in a significant increase in p53 acetylation. Inhibition of p53 acetylation by C646 significantly alleviated ferroptosis in H9c2 myofibroblasts. To further explore the involvement of p53 acetylation in SIRT3-mediated ferroptosis, we crossed acetylated p53 mutant (p534KR) mice, which cannot activate ferroptosis, with SIRT3KO mice. SIRT3KO/p534KR mice exhibited a significant reduction in ferroptosis and less cardiac fibrosis compared to SIRT3KO mice. Furthermore, cardiomyocyte-specific knockout of SIRT3 (SIRT3-cKO) in mice resulted in a significant increase in ferroptosis and cardiac fibrosis. Treatment of SIRT3-cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) led to a significant reduction in ferroptosis and cardiac fibrosis. We concluded that SIRT3-mediated cardiac fibrosis was partly through a mechanism involving p53 acetylation-induced ferroptosis in myofibroblasts.

Sirt3-dependent regulation of mitochondrial oxidative stress and apoptosis contributes to the dysfunction of pancreatic islets after severe burns.

Severe burns are often complicated with hyperglycemia caused by mitochondrial oxidative stress-related pancreatic islet dysfunction. Silent information regulator of transcription 3 (Sirt3) can regulate mitochondrial oxidative stress. However, the role and mechanism of Sirt3 on islet function after severe burns remain unclear. Therefore, this study aimed to investigate whether Sirt3 played a role in both mitochondrial oxidative stress in islets and mediating islet function post severe burns. A mouse model of 30% total body surface area full-thickness burn and an in vitro MIN6 cell hypoxia model were established. Sirt3 KO mice were used to demonstrate further the role of Sirt3 in maintaining redox homeostasis and regulating islet function. Fasting blood glucose and glucose-stimulated insulin secretion (GSIS) were detected to assess the islet function. The levels of mitochondrial ROS and deacetylation, and the activities of Mn-SOD and IDH2 were measured to evaluate oxidative stress. The mitochondrial membrane potential (MMP)was detected and the apoptosis rate measured. In vitro MIN6 cells, the hypoxia treatment significantly reduced Sirt3 expression, resulting in increased deacetylation of Mn-SOD and IDH2, which further led to a higher level of mitochondrial ROS. In addition, hypoxia reduced MMP and increased apoptosis rate, which impaired GSIS eventually. Knockdown of Sirt3 caused similar alterations. The hypoxia-induced high level of mitochondrial ROS and apoptosis and impaired GSIS could be reversed by overexpression of Sirt3. Similarly, after severe burns, the expression of Sirt3 in islets decreased significantly with a high level of deacetylation of Mn-SOD, IDH2, mitochondrial ROS and apoptosis, and islet dysfunction. Oxidative stress and apoptosis also occurred in islets of Sirt3 KO mice, accompanied by islet dysfunction. Sirt3 and downstream signalling are critical in modulating the islet function post severe burns by regulating mitochondrial oxidative stress and apoptosis.

Mitochondrial brain proteome acetylation levels and behavioural responsiveness to amphetamine are altered in mice lacking Sirt3.

Post-translational modification of mitochondrial proteins represents one mechanism by which the functional activity of mitochondria can be regulated. In the brain, these modifications can influence the functional properties of different neural circuitries. Given that the sirtuin family member Sirt3 represents the primary protein deacetylase enzyme in mitochondria, we tested whether brain mitochondrial proteome acetylation would increase in male or female mice lacking Sirt3. Our results confirm that whole brain mitochondrial proteome acetylation levels are indeed elevated in both sexes of Sirt3-KO mice relative to controls. Consistently, we found the mitochondria of mouse embryonic fibroblast (MEF) cells derived from Sirt3-KO mice were smaller in size, and fewer in number than in wild-type MEFs, and that mitochondrial free calcium levels were elevated within the mitochondria of these cells. As protein acetylation can influence mitochondrial function, and changes in mitochondrial function have been linked to alterations in neural circuit function regulating motor activity and anxiety-like behavior, we tested whether Sirt3-deficient mice would display sensitized responsiveness to the stimulant amphetamine. Both male and female Sirt3-KO mice displayed hyper-locomotion and attenuated anxiety-like behavior in response to a dose of amphetamine that was insufficient to promote any behavioural responses in wild-type mice. Collectively, these results confirm that Sirt3 regulates mitochondrial proteome acetylation levels in brain tissue, and that the absence of Sirt3 increases the sensitivity of neural systems to amphetamine-induced behavioural responses.

SS31 Alleviates Pressure Overload-Induced Heart Failure Caused by Sirt3-Mediated Mitochondrial Fusion

Heart failure caused by pressure overload is one of the leading causes of heart failure worldwide, but its pathological origin remains poorly understood. It remains critical to discover and find new improvements and treatments for pressure overload-induced heart failure. According to previous studies, mitochondrial dysfunction and myocardial interstitial fibrosis are important mechanisms for the development of heart failure. The oligopeptide Szeto-Schiller Compound 31 (SS31) can specifically interact with the inner mitochondrial membrane and affect the integrity of the inner mitochondrial membrane. Whether SS31 alleviates pressure overload-induced heart failure through the regulation of mitochondrial fusion has not yet been confirmed. We established a pressure-overloaded heart failure mouse model through TAC surgery and found that SS31 can significantly improve cardiac function, reduce myocardial interstitial fibrosis, and increase the expression of optic atrophy-associated protein 1 (OPA1), a key protein in mitochondrial fusion. Interestingly, the role of SS31 in improving heart failure and reducing fibrosis is inseparable from the presence of sirtuin3 (Sirt3). We found that in Sirt3KO mice and fibroblasts, the effects of SS31 on improving heart failure and improving fibroblast transdifferentiation were disappeared. Likewise, Sirt3 has direct interactions with proteins critical for mitochondrial fission and fusion. We found that SS31 failed to increase OPA1 expression in both Sirt3KO mice and fibroblasts. Thus, SS31 can alleviate pressure overload-induced heart failure through Sirt3-mediated mitochondrial fusion. This study provides new directions and drug options for the clinical treatment of heart failure caused by pressure overload.

Sirt3 Protects Against Thoracic Aortic Dissection Formation by Reducing Reactive Oxygen Species, Vascular Inflammation, and Apoptosis of Smooth Muscle Cells.

Sirtuin3 (Sirt3) is a histone deacetylase involved in the regulation of many cellular processes. Sirt3 deficiency is known to increase oxidative stress. Reactive oxygen species (ROS) promote degradation of the extracellular matrix and vascular smooth muscle cell (VSMC) apoptosis. Reducing oxidative stress by Sirt3 overexpression could have therapeutic potential for limiting thoracic aortic dissection (TAD) development. We hypothesized that Sirt3 deficiency could increase the risk for TAD by decreasing ROS elimination and that Sirt3 overexpression (Sirt3OE) could provide an alternative option for TAD treatment. Mice with TAD had significantly lower Sirt3 expression than normal subjects. Sirt3 KO mice exhibit significantly increased TAD incidence rate and increased aortic diameters. Moreover, Sirt3 overexpression reduced Ang II-induced ROS production, NF-kB activation, and apoptosis in human aortic smooth muscle cells (HASMCs). Sirt3 overexpression attenuated aneurysm formation and decreased aortic expansion. In conclusion, our data showed that Sirt3 deficiency increases susceptibility to TAD formation by attenuating anti-ROS effects and increasing VSMC apoptosis and vascular inflammation.

Abstract IA-023: The role of sirtuins in radiation-induced cardiac dysfunction

Abstract Currently there is no way to reliably predict who is at higher or lower risk of cardiac toxicity from radiation, and the critical factors that alter radiation-induced heart disease are not completely clear. The heart produces and uses the most energy of all organs, with >90% of its energy produced via mitochondrial respiration. To maintain this energy output, ~30% of the heart volume is made up of mitochondria in cardiomyocytes. Such a high metabolic rate produces increased levels of reactive oxygen species (ROS), which have the potential to cause serious oxidative damage to the heart. Antioxidant enzymes such as superoxide dysmutases (SODs), catalase, and glutathione peroxidase can help to protect against ROS-mediated damage by converting superoxide to hydrogen peroxidase, and then hydrogen peroxide to water. Oxidative stressors, including therapeutic radiation therapy, have been shown to lead to long-term effects on mitochondrial function, as measured by excess formation of ROS including superoxide, mitochondrial-related gene expression, and altered mitochondrial morphology and functions such as mitochondrial respiration, pyruvate metabolism, and ion and lipid transport. While a potential link between aging-related signaling pathways, metabolic homeostasis, and radiation-induced normal tissue damage has recently been suggested, rigorous mechanistic data to support this intriguing idea has been limited. Mitochondrial detoxification pathways, which are dysregulated in normal tissues by radiation, lead to increased ROS levels and the disruption of mitochondrial reparative pathways. SIRT3 is one of three mitochondrial-located sirtuin proteins. SIRT3 is the primary deacetylase of mitochondrial proteins, and its deacetylase activity plays a key role in maintaining mitochondrial energy metabolism equilibrium and defending against cellular damage that occurs with aging. Lysine acetylation is an important post-translational modification that can regulate the function of mitochondrial proteins. The acetylation status of key mitochondrial detoxification proteins, which are Sirt3 deacetylation targets, may be dysregulated in normal tissue following radiation exposure. Loss of SIRT3 can dysregulate mitochondrial detoxification and metabolism, leading to the accumulation of cellular damage. Sirt3 KO mice demonstrate enhanced mitochondrial dysfunction, and Sirt3 KO mice exhibit enhanced doxorubicin- and aortic constriction-induced cardiac hypertrophy. In addition, Sirt3 KO MEFs and livers are more sensitive to radiation and exhibit increased mitochondrial superoxide levels. However, the role of sirtuins in radiation-induced cardiac injury has not been examined. Here we review data on the potential role of sirtuins, and in particular Sirt3, on radiation-induced cardiac function. Citation Format: Carmen Bergom. The role of sirtuins in radiation-induced cardiac dysfunction [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-023.

Application of a real-ambient fine particulate matter exposure system on different animal models

Simulation of fine particulate matter (PM2.5) exposure is essential for evaluating adverse health effects. In this work, an ambient exposure system that mimicked real atmospheric conditions was installed in Taiyuan, China to study impacts of chronic PM2.5 exposure on adult and aged mice as well as Sirtuin3 knockout (Sirt3 KO) mice and wild-type (WT) mice. The real-ambient exposure system eliminated the possible artificial effects caused from exposure experiments and maintained the physiochemical characteristics of PM2.5. The case studies indicated that aged mice exhibited apparent heart dysfunction involving increased heart rate and decreased blood pressure after 17-week of real-ambient PM2.5 exposure. Meanwhile, 15-week of real-ambient PM2.5 exposure decreased the heart rate and amounts of associated catecholamines to induce heart failure in Sirt3 KO mice. Additionally, the increased pro-inflammatory cytokines and decreased platelet related indices suggested that inflammation occurred. The changes of biomarkers detected by targeted metabolomics confirmed metabolic disorder in WT and Sirt3 KO mice after exposed to real-ambient PM2.5. These results indicated that the real-ambient PM2.5 exposure system could evaluate the risks of certain diseases associated with air pollution and have great potential for supporting the investigations of PM2.5 effects on other types of rodent models.

SIRT3 promotes auditory function in young adult FVB/nJ mice but is dispensable for hearing recovery after noise exposure.

Noise-induced hearing loss (NIHL) affects millions of people worldwide and presents a large social and personal burden. Pharmacological activation of SIRT3, a regulator of the mitochondrial oxidative stress response, has a protective effect on hearing thresholds after traumatic noise damage in mice. In contrast, the role of endogenously activated SIRT3 in hearing recovery has not been established. Here we tested the hypothesis that SIRT3 is required in mice for recovery of auditory thresholds after a noise exposure that confers a temporary threshold shift (TTS). SIRT3-specific immunoreactivity is present in outer hair cells, around the post-synaptic regions of inner hair cells, and faintly within inner hair cells. Prior to noise exposure, homozygous Sirt3-KO mice have slightly but significantly higher thresholds than their wild-type littermates measured by the auditory brainstem response (ABR), but not by distortion product otoacoustic emissions (DPOAE). Moreover, homozygous Sirt3-KO mice display a significant reduction in the progression of their peak 1 amplitude at higher frequencies prior to noise exposure. After exposure to a single sub-traumatic noise dose that does not permanently reduce cochlear function, compromise cell survival, or damage synaptic structures in wild-type mice, there was no difference in hearing function between the two genotypes, measured by ABR and DPOAE. The numbers of hair cells and auditory synapses were similar in both genotypes before and after noise exposure. These loss-of-function studies complement previously published gain-of-function studies and help refine our understanding of SIRT3's role in cochlear homeostasis under different damage paradigms. They suggest that SIRT3 may promote spiral ganglion neuron function. They imply that cellular mechanisms of homeostasis, in addition to the mitochondrial oxidative stress response, act to restore hearing after TTS. Finally, we present a novel application of a biomedical statistical analysis for identifying changes between peak 1 amplitude progressions in ABR waveforms after damage.

SIRT3 Ablation Deteriorates Obesity-Related Cardiac Remodeling by Modulating ROS-NF-κB-MCP-1 Signaling Pathway.

Obesity and the associated complications are a major public health issue as obesity incidence increases yearly, worldwide. Effects of obesity on heart failure have been reported previously. Obesity-related cardiac remodeling includes structural and functional dysfunctions, in which cardiac inflammation and fibrosis play a key role. The main mitochondrial deacetylase, SIRT3 participates in numerous cellular processes; however, its role in obesity-related cardiac remodeling remains unclear. In our study, high-fat diet (HFD) feeding induced downregulation of SIRT3 protein level in mice. SIRT3-KO mice fed on HFD exhibited higher cardiac dysfunction and cardiac remodeling compared with the wild-type controls. Further study revealed increases in collagen accumulation and inflammatory cytokine expression including MCP-1, IL-6, TGF-β, TNF-α in mice fed on HFD compared with chow diet, with higher levels observed in SIRT3-KO mice. Furthermore, significantly high levels of cardiac MCP-1 expression and macrophage infiltration, and ROS generation and activated NF-κB were observed in HFD-fed SIRT3-KO mice. We presumed that SIRT3 ablation-mediated MCP-1 upregulation is attributed to ROS-NF-κB activation. Thus, we concluded that SIRT3 prevents obesity-related cardiac remodeling by attenuating cardiac inflammation and fibrosis, through modulation of ROS-NF-κB-MCP-1 pathway.

Sirtuin 3 is essential for hypertension‐induced cardiac fibrosis via mediating pericyte transition

Hypertension is the key factor for the development of cardiac fibrosis and diastolic dysfunction. Our previous study showed that knockout of sirtuin 3 (SIRT3) resulted in diastolic dysfunction in mice. In the present study, we explored the role of SIRT3 in angiotensin II (Ang‐II)–induced cardiac fibrosis and pericyte‐myofibroblast transition. NG2 tracing reporter NG2‐DsRed mouse was crossed with wild‐type (WT) mice and SIRT3KO mice. Cardiac function, cardiac fibrosis and reactive oxygen species (ROS) were measured. Mice infused with Ang‐II for 28 days showed a significant reduction of SIRT3 expression in the mouse hearts. Knockout of SIRT3 sensitized Ang‐II‐induced elevation of isovolumic relaxation time (IVRT) and reduction of ejection fraction (EF) and fractional shortening (FS). Ang‐II‐induced cardiac fibrosis, capillary rarefaction and hypertrophy were further enhanced by knockout of SIRT3. NG2 pericyte tracing reporter mice infused with Ang‐II had a significantly increased number of NG2‐DsRed pericyte in the heart. Knockout of SIRT3 further enhanced Ang‐II‐induced increase of pericytes. To examine pericyte‐myofibroblast/fibroblast transition, DsRed pericytes were co‐stained with FSP‐1 and α‐SMA. Ang‐II infusion led to a significant increase in numbers of DsRed+/FSP‐1+ and DsRed+/α‐SMA+ cells, while SIRT3KO further developed pericyte‐myofibroblast/fibroblast transition. In addition, knockout of SIRT3 promoted Ang‐II‐induced NADPH oxidase‐derived ROS formation together with increased expression of transforming growth factor beta 1 (TGF‐β1). We concluded that Ang‐II induced cardiac fibrosis partly by the mechanisms involving SIRT3‐mediated pericyte‐myofibroblast/fibroblast transition and ROS‐TGF‐β1 pathway.

Abstract P180: Deficiency of Sirtuin 3 Sensitizes Angiotensin II-Induced Cardiac Pericyte-Fibroblast Transition and Diastolic Dysfunction via Iron Overloading

Hypertension is the key factor for the development of cardiac fibrosis and diastolic dysfunction. Our previous study showed that knockout of SIRT3 resulted in diastolic dysfunction in mice. In present study, we aim to explore the role of SIRT3 in pericyte recruitment and cardiac fibrosis in response to hypertension. NG2 + tracing reporter NG2DsREDBAC mouse was crossed with SIRT3KO mice. Mice were infused with Ang-II (1000ng/kg/min) for 28 days, blood pressure, coronary flow reserve (CFR) and diastolic function (IVRT and MPI) were measured pre- and post- Ang-II treatment. Cardiac fibrosis and iron content were measured by Masson and Prussian staining. Pericytes and fibroblasts were measured by co-immunostaining. The expression of heme oxygenase-1 (HO-1), ferroportin (FPN), p47 phox , and transforming growth factor-β1(TFG-β1) was analyzed by western blots. Infusion of Ang-II resulted in a significant increase in blood pressure compared to basal levels in WT mice (MAP 94± 2 mHg vs 130±1.7 mHg, n=7, p<0.05); whereas knockout of SIRT3 exacerbated Ang-II-induced increase in blood pressure (MAP WT 130±1.7 mHg vs SIRT3KO 145 ± 2.1 mHg, n=7, p<0.05). Furthermore, knockout of SIRT3 sensitized Ang-II-induced reduction of CFR and elevation of IVRT and MPI in mice. Using NG2 pericyte tracing reporter mice, we found that mice infusion with Ang-II resulted in a significant increase in NG2 + pericyte recruitment in the heart. Knockout of SIRT3 enhanced Ang-II-induced NG2 + pericyte recruitment. Knockout of SIRT3 further enhanced Ang-II-induced cardiac fibrosis as well as fibrotic markers FSP1 and a-SMA staining. To examine whether pericytes differentiate into fibroblast, NG2 + pericytes were co-stained with FSP-1 + . Ang-II infusion led to a significant increase in NG2 + /FSP-1 + ; while knockout of SIRT3 further increased numbers of NG2 + /FSP-1 + in the heart. Mechanistically, knockout of SIRT3 increased iron staining in the heart. This was accompanied by a significant increase in the expression of HO-1, FPN, p47 phox , ROS, and TGF-β as compared to Ang-II treatment. Our study suggests that deficiency of SIRT3 may promote Ang-II-induced cardiac fibrosis via pericyte-fibroblast transition and by Fe2 + -ROS-TGF-β pathway.

SIRT3-MEDIATED MACROPHAGE PYRUVATE DEHYDROGENASE ACTIVITY IMPROVES PERIVASCULAR ADIPOSE TISSUE REMODELING IN HYPERTENSIVE MICE

Objective: Infiltrated macrophages are a dominant population in the perivascular adipose tissue (PVAT) microenvironment and actively promote PVAT remodeling in hypertensive mice. However, the molecular mechanism underlying the role of macrophages in initiating metabolic inflammation remains uncertain. Here, we investigate whether Situin-3(SIRT3) is determinant in macrophage activation. Design and method: 10-week-old male WT, SIRT3 KO, and NLRP3 KO mice were infused with Angiotension II (Ang II, 1000ng/kg/min) or normal saline (NS) for 7 days. The morphological and functional changes of thoracic aorta and PVAT were determined by histological analysis and immunofluorescence staining. In vitro studies were performed on bone marrow-derived macrophages (BMDMs) and brown adipocytes isolated from WT or SIRT3 KO mice. Results: We report that Ang II accelerates PVAT inflammation and fibrosis in SIRT3-deficient mice. This effect is associated with attenuating browning markers and lipid droplet-associated protein expression. Immunofluorescence analysis confirmed that there is an increase in NLRP3 inflammasome and secretion of interleukin-1β (IL-1β). In vitro studies indicate that BMDMs from SIRT3 KO mice induce interleukin-IL-1β production by shifting metabolic phenotype from oxidative phosphorylation towards glycolysis. Mechanically, SIRT3 deacetylates and activates pyruvate dehydrogenase E1 alpha (PDHE1a) at lysine 83, loss of SIRT3 lead to decreased PDH activity, and accumulation of pyruvate and lactate metabolites. The PDHK inhibitor dichloroacetate (DCA) suppresses both pro-IL-1β maturation and caspase-1 activation in macrophages in response to Ang II. The blockade of IL-1β from macrophage into brown adipocytes restores expression of brown adipocyte specific markers. Coincidentally, NLRP3 KO mice exhibited a reduction in infiltrated macrophages in PVAT, while partially rescuing brown adipocytes mitochondrial function and collagen accumulation. Conclusions: Taken together, these findings reveal that SIRT3-mediated activity of PDHE1a inhibits macrophage activation. Pharmacological targeting of glycolysis metabolism may therefore represent an effective approach.

1973-P: Deficiency of Protein Deacetylase Sirt3 Enhances Acetylation of Histone H3 and H4 as to Affect Glucose Metabolism through Regulation of Dipeptidyl Peptidase-4 in Mice

Background: Sirt3 is a NAD-dependent deacetylase that regulates lipid metabolism in mice and is related to longevity through modification of epigenome. Sirt3-deficient knockout (KO) mice fed a high-fat diet have been shown to develop obesity and metabolic syndrome-like phenotype. However, the precise mechanism by which they cause glucose intolerance has not been known. To reveal the regulatory mechanism of glucose metabolism through Sirt3, the metabolic parameters in the Sirt3-KO mice were examined. Objective and Methods: The histone acetylation levels in various organs of the Sirt3-KO mice were examined by immunostaining of acetylated histone H3 (H3Ac) and H4 (H4Ac). Blood glucose levels in the Sirt3-KO mice after glucose loading were examined, associated with blood glucagon-like peptide-1 (GLP-1) levels and dipeptidyl peptidase-4 (DPP-4) activity. Gene expressions which were associated with the expressions and functions of GLP-1 and DPP-4 were quantified by qPCR. Results: The Sirt3-KO mice were subjected to an oral glucose tolerance test (OGTT), together with an intraperitoneal glucose tolerance test (ipGTT). OGTT revealed an increase in the blood glucose levels in the KO mice, although ipGTT did not show the increase after the glucose loading. The blood GLP-1 and insulin levels decreased, and the DPP-4 activity increased significantly in the KO mice. qPCR analysis revealed a predominance of Sirt3 expression in the intestinal tract in the wild type mice. The immunostaining of H3Ac and H4Ac showed enhanced intensity in the small intestine and colon, and in the vascular endothelial cells in the KO mice. Conclusion: These results suggest that Sirt3 deficiency impairs the incretin effect by a suppression of the blood GLP-1 levels and causes glucose intolerance. Enhanced acetylation of histone H3 and H4 in the KO mice might relate to the increase in the DPP-4 activity, which lead to the decrease in the GLP-1 levels. Disclosure A. Uto: None. K. Miyashita: Research Support; Self; Johnson & Johnson, Merck & Co., Inc., Novo Nordisk Inc.

36-OR: Energy Stress Restores Muscle Insulin Action in High-Fat (HF)-Fed SIRT3 KO Mice

SIRT3 is a mitochondrial deacetylase that is necessary for insulin-stimulated muscle glucose uptake (MGU) in the setting of overnutrition, and its action is redox state-dependent (NAD/NADH). Energy stress induced by AICAR administration increases muscle insulin action and glucose uptake while enhancing the control of redox state. We hypothesized that acute energy stress would exacerbate the impairment in insulin-stimulated MGU in high fat-fed (HF) SIRT3 KO mice. To test this hypothesis, HF SIRT3 KO and WT littermates underwent 4h of either AICAR (1mg/kg/min) to induce an energetic stress or saline infusion followed by hyperinsulinemic (4mU/kg/min) euglycemic clamps. Experiments were conducted one week post jugular vein and carotid artery catheterization. Insulin clamps were combined with [3-3H] glucose to measure glucose fluxes and 2[14C]deoxyglucose to assess an index of MGU. AICAR significantly improved insulin sensitivity of liver, but not muscle of HF WT mice. Saline-SIRT3 KO mice were more insulin resistant than saline-WT mice, as glucose infusion rate (GIR) and MGU were reduced by ~50%. AICAR completely abolished the added insulin resistance in SIRT3 KO mice as GIR was similar to AICAR-WT. Consistent with this, AICAR increased muscle insulin-stimulated MGU in the SIRT3 KO mice to levels observed in WT mice. Results show that energy stress induced by AICAR infusion was sufficient to circumvent the defect in insulin-stimulated glucose uptake induced by SIRT3 KO, possibly by rescuing redox handling in skeletal muscle. Disclosure L. Lantier: None. C.C. Hughey: None. D. Wasserman: None. Funding National Institutes of Health (DK54902)

SIRT3 controls brown fat thermogenesis by deacetylation regulation of pathways upstream of UCP1

ObjectiveBrown adipose tissue (BAT) is important for thermoregulation in many mammals. Uncoupling protein 1 (UCP1) is the critical regulator of thermogenesis in BAT. Here we aimed to investigate the deacetylation control of BAT and to investigate a possible functional connection between UCP1 and sirtuin 3 (SIRT3), the master mitochondrial lysine deacetylase. MethodsWe carried out physiological, molecular, and proteomic analyses of BAT from wild-type and Sirt3KO mice when BAT is activated. Mice were either cold exposed for 2 days or were injected with the β3-adrenergic agonist, CL316,243 (1 mg/kg; i.p.). Mutagenesis studies were conducted in a cellular model to assess the impact of acetylation lysine sites on UCP1 function. Cardiac punctures were collected for proteomic analysis of blood acylcarnitines. Isolated mitochondria were used for functional analysis of OXPHOS proteins. ResultsOur findings showed that SIRT3 absence in mice resulted in impaired BAT lipid use, whole body thermoregulation, and respiration in BAT mitochondria, without affecting UCP1 expression. Acetylome profiling of BAT mitochondria revealed that SIRT3 regulates acetylation status of many BAT mitochondrial proteins including UCP1 and crucial upstream proteins. Mutagenesis work in cells suggested that UCP1 activity was independent of direct SIRT3-regulated lysine acetylation. However, SIRT3 impacted BAT mitochondrial proteins activities of acylcarnitine metabolism and specific electron transport chain complexes, CI and CII. ConclusionsOur data highlight that SIRT3 likely controls BAT thermogenesis indirectly by targeting pathways upstream of UCP1.

The absence of SIRT3 and SIRT5 promotes the acetylation of lens proteins and improves the chaperone activity of α-crystallin in mouse lenses

Acetylation of lysine residues occurs in lens proteins. Previous studies have shown an improvement in the chaperone activity of αA-crystallin upon acetylation. Sirtuins are NAD+-dependent enzymes that can deacylate proteins. The roles of sirtuins in regulating the acetylation of lens proteins and their impacts on the function of α-crystallin are not known. Here, we detected sirtuin activity in mouse lenses, and SIRT3 and SIRT5 were present primarily in the mitochondria of cultured primary mouse lens epithelial cells. Western blotting showed higher levels of protein acetylation in the lenses of SIRT3 KO and SIRT5 KO mice than in lenses of WT mice. Mass spectrometry analyses revealed a greater number of acetylated lysine residues in α-crystallin isolated from the SIRT3 and SIRT5 KO lenses than from WT lenses. α-Crystallin isolated from SIRT3 and SIRT5 KO lenses displayed a higher surface hydrophobicity and higher chaperone activity than the protein isolated from WT lenses. Thus, SIRTs regulate the acetylation levels of crystallins in mouse lenses, and acetylation in lenses enhances the chaperone activity of α-crystallin.

Sirtuin 3 deficiency aggravates contrast-induced acute kidney injury

BackgroundSirtuin 3 (Sirt3) is a key regulator of energy metabolism and oxidative stress. To investigate the role of Sirt3 in contrast-induced acute kidney injury (CIAKI), we established the model both in vivo and in vitro to explore the potential mechanisms.MethodsIn vivo, we established CIAKI models in wild-type (WT) and Sirt3-knockout (Sirt3-KO) mice. Blood urea nitrogen (BUN) and serum creatinine (Scr) were detected by enzyme-linked immunosorbent assay, Glomerular Filtration Rate (GFR) and creatinine clearance were also investigated. We detected the production of reactive oxygen species (ROS) via 2′7′-dichlorodihydro-fluorescein diacetate. The expressions of Sirt3, oxidative stress and apoptosis related markers (MnSOD, Catalase, Acetyl-MnSOD K68, Nox4, Bax, Bcl-2 and Caspase3) were measured and analyzed. In addition, we observed the effect of nicotinamide riboside (NR) on CIAKI in WT and Sirt3-KO mice. In vitro, Sirt3 was knocked out by siRNA transfection method in HK-2 cells. Sirt3, ROS, oxidative stress and apoptosis markers in HK-2 cells were also measured.ResultsOur data demonstrated that the levels of Scr and BUN in Sirt3-KO mice were increased while the levels of the GFR and creatinine clearance were decreased in CIAKI mice. In Sirt3-KO or siRNA groups, the activities of MnSOD and Catalase were markedly down-regulated. Also, the expression of Caspase3 were markedly increased and the ratio of Bcl-2/Bax was decreased, while the ROS level was increased in Sirt3 deficiency groups. NR ameliorated CIAKI in WT mice but not in Sirt3-KO mice.ConclusionOur results suggest that Sirt3 deficiency aggravates contrast-induced acute kidney injury. Sirt3 is critical in NR-mediated renoprotection in CIAKI.

Abstract 009: Regulatory Role of Endothelial Sirt3 on Blood Pressure and Diastolic Dysfunction in a Female Mice

Heart failure with preserved ejection fraction (HFpEF) accounts for almost one half of the heart failure population which is characterized by a diastolic dysfunction. HFpEF is strongly associated with advanced age and metabolic comorbidities as well as endothelial dysfunction. Sirt3 emerged as a protein of particular interest to the aging due to its association with long lifespans in humans. SIRT3KO mice have decreased time to development of age-related diseases. Diastolic dysfunction is increasingly prevalent in aged women. Clinical studies have shown that obese and diabetic women are more likely to develop diastolic dysfunction than their male counterparts. Our study showed that ablation of endothelial Sirt3 led to diastolic dysfunction in male mice. However, the role of endothelial SIRT3 deficiency on blood pressure and diastolic function in female mice remains to be investigated. In the present study, we demonstrate that ablation of endothelial SIRT3 in females led to an increase in blood pressure as compared with control SIRT3 LoxP female mice. Diastolic function measurement also showed that IVRT and MPI were significantly increased whereas E’/A’ ratio was reduced in the SIRT3 EC KO female mice. To further investigate the regulatory role of endothelial SIRT3 on blood pressure and diastolic dysfunction in metabolic stress, SIRT3 EC KO female mice and SIRT3 LoxP female mice were fed with normal diet and high-fat diet (HFD) for 16 weeks. Knockout of endothelial SIRT3 resulted in an increased blood pressure in female mice fed with HFD. Intriguingly, Sirt3 EC KO female mice + HFD exhibited impaired coronary flow reserve (CFR) and more serve diastolic dysfunction as evidence elevated IVRT as compared with control SIRT3 LoxP female mice + HFD. In addition, female SIRT3 EC KO mice had higher blood pressure and diastolic dysfunction as compared to male SIRT3 EC KO mice. Moreover, female SIRT3 EC KO mice + HFD had an impaired CFR and diastolic dysfunction as compared to male SIRT3 EC KO mice + HFD. These results implicate a sex different role of endothelial SIRT3 in regulating blood pressure and diastolic function in mice. Deficiency of endothelial SIRT3 may be responsible for a diastolic dysfunction in aging female.