Uncoupling Protein 1 Knockout Research Articles

Changes in development and function of skeletal muscle in UCP-1 knockout mice

Changes in development and function of skeletal muscle in UCP-1 knockout mice

UCP2 knockout exacerbates sepsis-induced intestinal injury by promoting NLRP3-mediated pyroptosis

Sepsis-induced intestinal injury is a common complication that increases the morbidity and mortality associated with sepsis. UCP2, a mitochondrial membrane protein, is involved in numerous cellular processes, including metabolism, inflammation, and pyroptosis. According to our previous studies, UCP2 expression increases in septic intestinal tissue. However, its function in intestinal damage is not known. This work investigated UCP2′s role in intestinal injury caused by sepsis. A sepsis mouse model was established in wild-type and UCP2-knockout (UCP2-KO) animals using cecal ligation and puncture (CLP). MCC950, an NLRP3 inflammasome inhibitor, was injected intraperitoneally 3 h before CLP surgery. Overall, significantly higher levels of UCP2 were observed in the intestines of septic mice. UCP2-KO mice subjected to CLP exhibited exacerbated intestinal damage, characterized by enhanced mucosal erosion, inflammatory cell infiltration, and increased intestinal permeability. Furthermore, UCP2 knockout significantly increased oxidative stress, inflammation, and pyroptosis in the CLP mouse intestines. Interestingly, MCC950 not only inhibited pyroptosis but also reversed inflammation, oxidative stress as well as damage to intestinal tissues as a result of UCP2 knockout. Our results highlighted the protective functions of UCP2 in sepsis-associated intestinal injury through modulation of inflammation and oxidative stress via NLRP3 inflammasome-induced pyroptosis.

Mitochondrial Uncoupling Protein-2 Ameliorates Ischemic Stroke by Inhibiting Ferroptosis-Induced Brain Injury and Neuroinflammation.

Ischemic stroke is a devastating disease in which mitochondrial damage or dysfunction substantially contributes to brain injury. Mitochondrial uncoupling protein-2 (UCP2) is a member of the UCP family, which regulates production of mitochondrial superoxide anion. UCP2 is reported to be neuroprotective for ischemic stroke-induced brain injury. However, the molecular mechanisms of UCP2 in ischemic stroke remain incompletely understood. In this study, we investigated whether and how UCP2 modulates neuroinflammation and regulates neuronal ferroptosis following ischemic stroke in vitro and in vivo. Wild-type (WT) and UCP2 knockout (Ucp2-/-) mice were subjected to middle cerebral artery occlusion (MCAO). BV2 cells (mouse microglial cell line) and HT-22 cells (mouse hippocampal neuronal cell line) were transfected with small interfering (si)-RNA or overexpression plasmids to knockdown or overexpress UCP2 levels. Cells were then exposed to oxygen-glucose deprivation and reoxygenation (OGD/RX) to simulate hypoxic injury in vitro. We found that UCP2 expression was markedly reduced in a time-dependent manner in both in vitro and in vivo ischemic stroke models. In addition, UCP2 was mainly expressed in neurons. UCP2 deficiency significantly enlarged infarct volumes, aggravated neurological deficit scores, and exacerbated cerebral edema in mice after MCAO. In vitro knockdown of Ucp2 and in vivo genetic depletion of Ucp2 (Ucp2-/- mice) increased neuronal ferroptosis-related indicators, including Fe2+, malondialdehyde, glutathione, and lipid peroxidation. Overexpression of UCP2 in neuronal cells resulted in reduced ferroptosis. Moreover, knockdown of UCP2 exacerbated neuroinflammation in BV2 microglia and mouse ischemic stroke models, suggesting that endogenous UCP2 inhibits neuroinflammation following ischemic stroke. Upregulation of UCP2 expression in microglia appeared to decrease the release of pro-inflammatory factors and increase the levels of anti-inflammatory factors. Further investigation showed that UCP2 deletion inhibited expression of AMPKα/NRF1 pathway-related proteins, including p-AMPKα, t-AMPKα, NRF1, and TFAM. Thus, UCP2 protects the brain from ischemia-induced ferroptosis by activating AMPKα/NRF1 signaling. Activation of UCP2 represents an attractive strategy for the prevention and treatment of ischemic stroke.

Uncoupling Protein 2 Alleviates Myocardial Ischemia/Reperfusion Injury by Inhibiting Cardiomyocyte Ferroptosis

Introduction: Following our recent finding that Ucp2 knockout promotes ferroptosis, we aimed to examine whether UCP2 alleviates myocardial ischemia/reperfusion injury (MI/RI) by inhibiting ferroptosis. Methods: The left anterior descending coronary arteries of wild-type and Ucp2−/− C57BL/6 mice were ligated for 30 min and reperfused for 2 h to establish an MI/RI model. The effects of UCP2 on ferroptosis and MI/RI were determined by echocardiography, 2,3,5-triphenylttrazolium chloride staining, hematoxylin-eosin staining, Masson’s trichrome staining, Sirius red staining, and analysis of myocardial injury markers and ferroptosis indicators. Ferrostatin-1 (Fer-1) and erastin (Era) were used to investigate whether UCP2 alleviated MI/RI by inhibiting ferroptosis and the molecular mechanism. Results: UCP2 was upregulated in the MI/RI model in WT mice. Deletion of Ucp2 exacerbated ferroptosis, altered the expression levels of multiple ferroptosis-related genes, and significantly exacerbated MI/RI. Knockout of Ucp2 promoted ferroptosis induced by Era and inhibited the antiferroptotic effects of Fer-1. Knockout of Ucp2 activated the p53/TfR1 pathway to exacerbate ferroptosis. Conclusion: Our results showed that UCP2 inhibited ferroptosis in MI/RI, which might be related to regulation of the p53/TfR1 pathway.

Oxaloacetic acid induces muscle energy substrate depletion and fatigue by JNK-mediated mitochondrial uncoupling.

Fatigue is a common phenomenon closely related to physical discomfort and numerous diseases, which is severely threatening the life quality and health of people. However, the exact mechanisms underlying fatigue are not fully characterized. Herein, we demonstrate that oxaloacetic acid (OAA), a crucial tricarboxylic acid cycle intermediate, modulates the muscle fatigue. The results showed that serum OAA level was positively correlated with fatigue state of mice. OAA-treated induced muscle fatigue impaired the exercise performance of mice. Mechanistically, OAA increased the c-Jun N-terminal kinase (JNK) phosphorylation and uncoupling protein 2 (UCP2) levels in skeletal muscle, which led to decreased energy substrate and enhanced glycolysis. On the other hand, OAA boosted muscle mitochondrial oxidative phosphorylation uncoupled with energy production. In addition, either UCP2 knockout or JNK inhibition totally reversed the effects of OAA on skeletal muscle. Therein, JNK mediated UCP2 activation with OAA-treated. Our studies reveal a novel role of OAA in skeletal muscle metabolism, which would shed light on the mechanism of muscle fatigue and weakness.

Mitochondrial network adaptations of microglia reveal sex-specific stress response after injury and UCP2 knockout

Mitochondrial networks remodel their connectivity, content, and subcellular localization to support optimized energy production in conditions of increased environmental or cellular stress. Microglia rely on mitochondria to respond to these stressors, however our knowledge about mitochondrial networks and their adaptations in microglia invivo is limited. Here, we generate a mouse model that selectively labels mitochondria in microglia. We identify that mitochondrial networks are more fragmented with increased content and perinuclear localization invitro vs. invivo. Mitochondrial networks adapt similarly in microglia closest to the injury site after optic nerve crush. Preventing microglial UCP2 increase after injury by selective knockout induces cellular stress. This results in mitochondrial hyperfusion in male microglia, a phenotype absent in females due to circulating estrogens. Our results establish the foundation for mitochondrial network analysis of microglia invivo, emphasizing the importance of mitochondrial-based sex effects of microglia in other pathologies.

Temperature-Dependent Effects of Eicosapentaenoic Acid (EPA) on Browning of Subcutaneous Adipose Tissue in UCP1 Knockout Male Mice.

Uncoupling protein 1 (UCP1) plays a central role in thermogenic tissues by uncoupling cellular respiration to dissipate energy. Beige adipocytes, an inducible form of thermogenic cells in subcutaneous adipose tissue (SAT), have become a major focus in obesity research. We have previously shown that eicosapentaenoic acid (EPA) ameliorated high-fat diet (HFD)-induced obesity by activating brown fat in C57BL/6J (B6) mice at thermoneutrality (30 °C), independently of UCP1. Here, we investigated whether ambient temperature (22 °C) impacts EPA effects on SAT browning in wild-type (WT) and UCP1 knockout (KO) male mice and dissected underlying mechanisms using a cell model. We observed resistance to diet-induced obesity in UCP1 KO mice fed HFD at ambient temperature, with significantly higher expression of UCP1-independent thermogenic markers, compared to WT mice. These markers included the fibroblast growth factor 21 (FGF21) and sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b), suggesting the indispensable role of temperature in beige fat reprogramming. Surprisingly, although EPA induced thermogenic effects in SAT-derived adipocytes harvested from both KO and WT mice, EPA only increased thermogenic gene and protein expression in the SAT of UCP1 KO mice housed at ambient temperature. Collectively, our findings indicate that the thermogenic effects of EPA, which are independent of UCP1, occur in a temperature-dependent manner.

Oxaloacetate regulates mitochondrial complex II respiration in brown fat: dependence on UCP1 expression

Objective: Oxaloacetate (OAA) is a long-known potent inhibitor of succinate dehydrogenase (SDH), a phenomenon that has, however, received only limited attention. We previously found that skeletal muscle mitochondria incubated at low membrane potential (Δψ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein Δψ is intrinsically low, accumulate OAA in amounts sufficient to inhibit SDH (complex II) respiration. We proposed a mechanism wherein low Δψ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance we carried out studies of mice with inherently different levels of IBAT mitochodnrial inner membrane potential. Hypothesis: Mice with differential expression of UCP1 would manifest Δψ-dependent differences in OAA-regulated mitochondrial respiration. Methodology: We examined several parameters in complex II and complex I energized IBAT mitochondria isolated from obesity prone C57BL/6J compared to obesity resistant 129SVE mice and from UCP1 knock-out (UCP1KO) compared to wildtype (WT) mice. Data were analyzed by unpaired, 2-tailed t-test and expressed as mean ± SE. Data: Isolated complex II (succinate)-energized IBAT mitochondria from 129SVE mice versus C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower Δψ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower Δψ, exhibited much lower respiration (AUC of respiration versus time, 19963 ± 1764 vs 36015 ± 2527, p = 0.0004, n = 6), greater OAA concentrations (22.9 ± 1.8 vs 12.1 ± 3.3, p = 0.016, n = 6), much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared to the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater Δψ, ROS, and NADH, but equal or greater O2 flux compared to WT mitochondria. GDP addition, compared to no GDP, increased Δψ and complex II respiration in WT mice associated with much less OAA. Respiration on complex I substrates for mitochondria of both strains of mice and for WT and UCP1KO mice followed the more classical dynamics of greater O2 flux at lower Δψ. Summary: These findings support the above-mentioned mechanism for OAA- and Δψ-dependent complex II respiration and support its physiologically relevance. Conclusions: Oxaloacetate regulates mitochondrial complex II dynamics in a manner dependent on inner membrane potential as intrinsically determined by mouse strain or genetic manipulation. NIH award, 1 R01 DK123043-01A1, VA Merit Review Award 2 I01 BX000285-06, and the Iowa Fraternal Order of the Eagles This is the full abstract presented at the American Physiology Summit 2023 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.

Matrine alleviates oxidative stress and ferroptosis in severe acute pancreatitis-induced acute lung injury by activating the UCP2/SIRT3/PGC1α pathway.

Acute lung injury (ALI) is one of the most serious complications of severe acute pancreatitis (SAP). Matrine is well known for its powerful antioxidant and antiapoptotic properties, although its specific mechanism of action in SAP-ALI is unknown. In this study, we examined the effects of matrine on SAP-associated ALIand the specific signaling pathways implicated in SAP-induced ALI, such as oxidative stress, the UCP2-SIRT3-PGC1α pathway, and ferroptosis. The administration of caerulein and lipopolysaccharide (LPS) to UCP2-knockout (UCP2-/-) and wild-type (WT) mice that were pretreated with matrine resulted in pancreatic and lung injury. Changes in reactive oxygen species (ROS) levels, inflammation, and ferroptosis in BEAS-2B and MLE-12 cells were measured following knockdown or overexpression and LPS treatment. Matrine inhibited excessive ferroptosis and ROS production by activating the UCP2/SIRT3/PGC1α pathway while reducing histological damage, edema, myeloperoxidase activity and proinflammatory cytokine expression in the lung. UCP2 knockout decreased the anti-inflammatory properties of matrine and reduced the therapeutic effects of matrine on ROS accumulation and ferroptosis hyperactivation. LPS-induced ROS production and ferroptosis activation in BEAS-2B cells and MLE-12 cells were further enhanced by knockdown of UCP2, but this effect was rescued by UCP2 overexpression. This study demonstrated that matrine reduced inflammation, oxidative stress, and excessive ferroptosis in lung tissue during SAP by activating the UCP2/SIRT3/PGC1α pathway, demonstrating its therapeutic potential in SAP-ALI.

Selective deficiency of UCP-1 and adropin may lead to different subtypes of anti-neutrophil cytoplasmic antibody-associated vasculitis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease that is prone to respiratory and renal failures. Its major target antigens are serine protease 3 (PR3) and myeloperoxidase (MPO), but the determinants of PR3 and MPO subtypes are still unclear. Uncoupling protein-1 (UCP-1) and adropin (Adr) regulate mutually and play an important role in endothelial cell injury. In this study, adropin and UCP-1 knockout (AdrKO and UCP-1-KO) models were established on the basis of C57BL/6 J mice. The results showed that UCP-1-KO and AdrKO mice similar to AAV: significant inflammatory cell infiltration, vascular wall damage, and erythrocyte extravasation. The pathological basis of AdrKO was that endothelial cells adhered and activated neutrophils to release MPO, and the core gene was peroxisome proliferator-activated receptor gamma (PPARG). However, UCP-1-KO induced PR3 release, and the accumulation and expression of tissue factor on the vascular wall, and the core gene was peroxisome proliferator-activated receptor delta (PPARD). The present study verified that the subtypes of AAV may be genetically different diseases and it also provide novel experimental evidence for clinical differentiation of the two subtypes.

Uncoupling protein 2 deficiency of non-cancerous tissues inhibits the progression of pancreatic cancer in mice

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. MethodsWe took advantage of a model of accelerated aging, uncoupling protein 2-deficient (Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type (WT) PDAC cells (cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. ResultsMeasurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ mRNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. ConclusionsUcp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.

UCP2 KO mice exhibit ameliorated obesity and inflammation induced by high-fat diet feeding

Uncoupling protein 2 (Ucp2) was first introduced as a member of Uncoupling protein family and a regulator of ROS formation; however, its role in adipose tissue is not fully understood. In the present study, we have investigated the role of Ucp2 against high-fat diet (HFD)-induced obesity in epididymal white adipose tissue (eWAT) and browning of inguinal white adipose tissue (iWAT). Diet-induced obesity is closely related to macrophage infiltration and the secretion of pro-inflammatory cytokines. Macrophages surround adipocytes and form a crown-like-structure (CLS). Some reports have suggested that CLS formation requires adipocyte apoptosis. After 12 weeks of HFD challenge, Ucp2 knockout (KO) mice maintained relatively lean phenotypes compared to wild-type (WT) mice. In eWAT, macrophage infiltration, CLS formation, and inflammatory cytokines were reduced in HFD KO mice compared to HFD WT mice. Surprisingly, we found that apoptotic signals were also reduced in the Ucp2 KO mice. Our study suggests that Ucp2 deficiency may prevent diet-induced obesity by regulating adipocyte apoptosis. However, Ucp2 deficiency did not affect the browning capacity of iWAT. [BMB Reports 2022; 55(10): 500-505].

Uncoupling Protein-1 Modulates Anxiety-Like Behavior in a Temperature-Dependent Manner.

A strong bidirectional link between metabolic and psychiatric disorders exists; yet, the molecular basis underlying this interaction remains unresolved. Here we explored the role of the brown adipose tissue (BAT) as modulatory interface, focusing on the involvement of uncoupling protein 1 (UCP-1), a key metabolic regulator highly expressed in BAT, in the control of emotional behavior. Male and female constitutive UCP-1 knock-out (KO) mice were used to investigate the consequences of UCP-1 deficiency on anxiety-related and depression-related behaviors under mild thermogenic (23°C) and thermoneutral (29°C) conditions. UCP-1 KO mice displayed a selective enhancement of anxiety-related behavior exclusively under thermogenic conditions, but not at thermoneutrality. Neural and endocrine stress mediators were not affected in UCP-1 KO mice, which showed an activation of the integrated stress response alongside enhanced fibroblast-growth factor-21 (FGF-21) levels. However, viral-mediated overexpression of FGF-21 did not phenocopy the behavioral alterations of UCP-1 KO mice and blocking FGF-21 activity did not rescue the anxiogenic phenotype of UCP-1 KO mice. No effects of surgical removal of the intrascapular BAT on anxiety-like behavior or FGF-21 levels were observed in either UCP-1 KO or WT mice. We provide evidence for a novel role of UCP-1 in the regulation of emotions that manifests as inhibitory constraint on anxiety-related behavior, exclusively under thermogenic conditions. We propose this function of UCP-1 to be independent of its activity in the BAT and likely mediated through a central role of UCP-1 in brain regions with converging involvement in energy and emotional control.SIGNIFICANCE STATEMENT In this first description of a temperature-dependent phenotype of emotional behavior, we propose uncoupling protein-1 (UCP-1), the key component of the thermogenic function of the brown adipose tissue, as molecular break controlling anxiety-related behavior in mice. We suggest the involvement of UCP-1 in fear regulation to be mediated through its expression in brain regions with converging roles in energy and emotional control. These data are important and relevant in light of the largely unexplored bidirectional link between metabolic and psychiatric disorders, which has the potential for providing insight into novel therapeutic strategies for the management of both conditions.

Sustained effect of habitual feeding time on daily rhythm of core body temperature in mice.

Background and aimCircadian clocks in most peripheral tissues are entrained mainly by feeding. Therefore, this study aimed to investigate whether the daily rhythm of core body temperature (CBT), including the effect of diet-induced thermogenesis, varies according to habitual feeding time.MethodsWild-type and uncoupling protein 1 (UCP1) knockout mice were fed only during the first 4 h (Breakfast group) or the last 4 h of the dark period (Dinner group) for 17 days. On day 18, both groups were fed twice for 2 h, at the same starting times. Locomotor activity and CBT were measured continuously during the experiment.ResultsOn day 18, CBT increased at the beginning of each feeding period, regardless of the group and strain. However, the CBT increase induced by the first meal decreased sharply in the Breakfast group and mildly in the Dinner group; the opposite was observed after the second meal. In UCP1 knockout, but not wild-type, mice, the total amount of CBT was significantly lower in the Dinner group than in the Breakfast group. These effects were mostly independent of the locomotor activity and food intake.ConclusionThese results reveal that the effect of habitual feeding time on the daily rhythm of CBT is sustained at least until the following day. These effects may be mediated by both UCP1-dependent and -independent mechanisms.

Endothelial UCP2 Is a Mechanosensitive Suppressor of Atherosclerosis.

Background:Inflamed endothelial cells (ECs) trigger atherogenesis, especially at arterial regions experiencing disturbed blood flow. UCP2 (Uncoupling protein 2), a key mitochondrial antioxidant protein, improves endothelium-dependent relaxation in obese mice. However, whether UCP2 can be regulated by shear flow is unknown, and the role of endothelial UCP2 in regulating inflammation and atherosclerosis remains unclear. This study aims to investigate the mechanoregulation of UCP2 expression in ECs and the effect of UCP2 on endothelial inflammation and atherogenesis.Methods:In vitro shear stress simulation system was used to investigate the regulation of UCP2 expression by shear flow. EC-specific Ucp2 knockout mice were used to investigate the role of UCP2 in flow-associated atherosclerosis.Results:Shear stress experiments showed that KLF2 (Krüppel-like factor 2) mediates fluid shear stress-dependent regulation of UCP2 expression in human aortic and human umbilical vein ECs. Unidirectional shear stress, statins, and resveratrol upregulate whereas oscillatory shear stress and proinflammatory stimuli inhibit UCP2 expression through altered KLF2 expression. KLF2 directly binds to UCP2 promoter to upregulate its transcription in human umbilical vein ECs. UCP2 knockdown induced expression of genes involved in proinflammatory and profibrotic signaling, resulting in a proatherogenic endothelial phenotype. EC-specific Ucp2 deletion promotes atherogenesis and collagen production. Additionally, we found endothelial Ucp2 deficiency aggravates whereas adeno-associated virus-mediated EC-Ucp2 overexpression inhibits carotid atherosclerotic plaque formation in disturbed flow-enhanced atherosclerosis mouse model. RNA-sequencing analysis revealed FoxO1 (forkhead box protein O1) as the major proinflammatory transcriptional regulator activated by UCP2 knockdown, and FoxO1 inhibition reduced vascular inflammation and disturbed flow-enhanced atherosclerosis. We showed further that UCP2 level is critical for phosphorylation of AMPK (AMP-activated protein kinase), which is required for UCP2-induced inhibition of FoxO1.Conclusions:Altogether, our studies uncover that UCP2 is novel mechanosensitive gene under the control of fluid shear stress and KLF2 in ECs. UCP2 expression is critical for endothelial proinflammatory response and atherogenesis. Therapeutic strategies enhancing UCP2 level may have therapeutic potential against atherosclerosis.

Pantothenate protects against obesity via brown adipose tissue activation.

Brown adipose tissue (BAT) is the primary site of adaptive thermogenesis, which is involved in energy expenditure and has received much attention in the field of obesity treatment. By screening a small-molecule compound library of drugs approved by the Food and Drug Administration, pantothenic acid was identified as being able to significantly upregulate the expression of uncoupling protein 1 (UCP1), a key thermogenic protein found in BAT. Pantothenate (PA) treatment decreased adiposity, reversed hepatic steatosis, and improved glucose homeostasis by increasing energy expenditure in C57BL/6J mice fed a high-fat diet. PA also significantly increased BAT activity and induced beige adipocytes formation. Mechanistically, the beneficial effects were mediated by UCP1 because PA treatment was unable to ameliorate obesity in UCP1 knockout mice. In conclusion, we identified PA as an effective BAT activator that can prevent obesity and may represent a promising strategy for the clinical treatment of obesity and related metabolic diseases.NEW & NOTEWORTHY PA treatment effectively and safely protected against obesity via the BAT-UCP1 axis. PA has therapeutic potential for treating obesity and type II diabetes.

Honokiol Ameliorates Post-Myocardial Infarction Heart Failure Through Ucp3-Mediated Reactive Oxygen Species Inhibition.

Post-myocardial infarction heart failure (post-MI HF) is one of the leading global causes of death, and current prevention and treatment methods still cannot avoid the increasing incidence. Honokiol (HK) has previously been reported to improve myocardial ischemia/reperfusion injury and reverse myocardial hypertrophy by activating Sirt1 and Sirt3. We suspect that HK may also have a therapeutic effect on post-MI HF. In this study, we aimed to investigate the efficacy and mechanism of HK in the treatment of post-MI HF. We found that HK inhibited myocardial reactive oxygen species (ROS) production, reduced myocardial fibrosis, and improved cardiac function in mice after MI. HK also reduced the abnormality of mitochondrial membrane potential (MMP) and apoptosis of cardiomyocytes caused by peroxide in neonatal cardiomyocytes. RNAseq results revealed that HK restored the transcriptome changes to a certain extent and significantly enhanced the expression of mitochondrial inner membrane uncoupling protein isoform 3 (Ucp3), a protein that inhibits the production of mitochondrial ROS, protects cardiomyocytes, and relieves heart failure after myocardial infarction (MI). In cardiomyocytes with impaired Ucp3 expression, HK cannot protect against the damage caused by peroxide. More importantly, in Ucp3 knockout mice, HK did not change the increase in the ROS level and cardiac function damage after MI. Taken together, our results suggest that HK can increase the expression of the cardioprotective protein Ucp3 and maintain MMP, thereby inhibiting the production of ROS after MI and ameliorating heart failure.

Lack of TRPV1 aggravates obesity-associated hypertension through the disturbance of mitochondrial Ca2+ homeostasis in brown adipose tissue

The combination of obesity and hypertension is associated with high morbidity and mortality; however, the mechanism underlying obesity-induced hypertension remains unclear. In this study, we detected the possible effects of TRPV1, a previously identified antihypertensive calcium (Ca2+) channel in adipose tissue, on the occurrence of obesity and hypertension in mice lacking UCP1, a spontaneously genetically manipulated obesity model, by generating TRPV1 and UCP1 double knockout mice. In these mice, obesity and hypertension appeared earlier and were more severe than in mice with the knockout of UCP1 or TRPV1 alone. The knockout of TRPV1 in UCP1 knockout mice further reduced functional brown adipose tissue (BAT) generation; decreased resting oxygen consumption, heat production, and locomotor activities; and was accompanied by severe mitochondrial respiratory dysfunction in BAT. Mechanistically, TRPV1, UCP1, and LETM1 acted as a complex to maintain an appropriate mitochondrial Ca2+ level, and TRPV1 knockout caused a compensatory increase in mitochondrial Ca2+ uptake via LETM1 activation. However, the compensatory response was blocked in UCP1−/− mice, resulting in dramatically reduced mitochondrial Ca2+ uptake and higher production of ATP and oxidative stress. This study provides in vivo evidence for the critical role of BAT mitochondrial Ca2+ homeostasis in obesity-associated hypertension and indicates that the TRPV1/UCP1/LETM1 complex may be an alternative intervention target.

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encodedgene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.

Uncoupling protein 1 knockout aggravates isoproterenol-induced acute myocardial ischemia via AMPK/mTOR/PPAR\u03b1 pathways in rats

Uncoupling protein 1 (UCP1) was found exclusively in the inner membranes of the mitochondria of brown adipose tissue (BAT). We found that UCP1 was also expressed in heart tissue and significantly upregulated in isoproterenol (ISO)-induced acute myocardial ischemia (AMI) rat model. The present study is to determine the underlying mechanism involved in the UCP1 upregulation in ISO-induced AMI rat model. The Ucp1−/− rats were generated by CRISPR-Cas9 system and presented decreased BAT volume. 2-months old Sprague Dawley (SD) wild-type (WT) and Ucp1−/− rats were treated with ISO intraperitoneally 30 mg/kg once a day for 3 consecutive days to establish AMI model. In saline group, the echocardiographic parameters, serum markers of myocardial injury cardiac troponin I (cTnI), creatine kinase isoenzyme MB (CK-MB), oxidant malondialdehyde (MDA), antioxidant superoxide dismutase (SOD) or fibrosis were comparable between WT and Ucp1−/− rats. ISO treatment induced worse left ventricle (LV) hypertrophy, myocardial fibrosis, increased higher cTnI, CK-MB and MDA and decreased lower SOD level in Ucp1−/− rats compared with that of WT rats. Ucp1−/− rats also presented lower myocardial phosphocreatine (PCr)/ATP-ratio, which demonstrated worse cardiac energy regulation defect. ISO treatment induced the phosphorylation of AMP-activated protein kinase (AMPK) activation, subsequently the phosphorylation of mammalian target of rapamycin (mTOR) inhibition and peroxisome proliferators-activated receptor α (PPARα) activation in WT rats, whereas activation of AMPK/mTOR/PPARα pathways significantly inhibited in Ucp1−/− rats. To sum up, UCP1 knockout aggravated ISO-induced AMI by inhibiting AMPK/mTOR/PPARα pathways in rats. Increasing UCP1 expression in heart tissue may be a cytoprotective therapeutic strategy for AMI.