Relaxin-3 Receptor (RXFP3) Signalling Mediates Stress-Related Alcohol Preference in Mice

Stressful life events are causally linked with alcohol use disorders (AUDs), providing support for a hypothesis that alcohol consumption is aimed at stress reduction. We have previously shown that expression of relaxin-3 mRNA in rat brain correlates with alcohol intake and that central antagonism of relaxin-3 receptors (RXFP3) prevents stress-induced reinstatement of alcohol-seeking. Therefore the objectives of these studies were to investigate the impact of Rxfp3 gene deletion in C57BL/6J mice on baseline and stress-related alcohol consumption. Male wild-type (WT) and Rxfp3 knockout (KO) (C57/B6J^{RXFP3TM1/DGen}) littermate mice were tested for baseline saccharin and alcohol consumption and preference over water in a continuous access two-bottle free-choice paradigm. Another cohort of mice was subjected to repeated restraint followed by swim stress to examine stress-related alcohol preference. Hepatic alcohol and aldehyde dehydrogenase activity was assessed in mice following chronic alcohol intake and in naive controls. WT and Rxfp3 KO mice had similar baseline saccharin and alcohol preference, and hepatic alcohol processing. However, Rxfp3 KO mice displayed a stress-induced reduction in alcohol preference that was not observed in WT littermates. Notably, this phenotype, once established, persisted for at least six weeks after cessation of stress exposure. These findings suggest that in mice, relaxin-3/RXFP3 signalling is involved in maintaining high alcohol preference during and after stress, but does not appear to strongly regulate the primary reinforcing effects of alcohol.     

A convenient method for europium‐labeling of a recombinant chimeric relaxin family peptide R3/I5 for receptor‐binding assays

Relaxin family peptides have important biological functions, and so far, four G‐protein‐coupled receptors have been identified as their receptors (RXFP1–4). A chimeric relaxin family peptide R3/I5, containing the B‐chain of relaxin‐3 and the A‐chain of INSL5, is a selective agonist for both RXFP3 and RXFP4. In a previous study, europium‐labeled R3/I5, as a nonradioactive and low‐background receptor‐binding tracer, was prepared through a chemical synthesis approach. In the present study, we established a convenient alternative approach for preparing the europium‐labeled R3/I5 tracer based on a recombinant R3/I5 designed to carry a solubilizing tag at the A‐chain N‐terminus and a pyroglutamate residue at the B‐chain N‐terminus. Because of the presence of a single primary amine moiety, the recombinant R3/I5 peptide was site‐specifically mono‐labeled at the A‐chain N‐terminus by a diethylenetriaminepentaacetic acid/europium moiety through a convenient one‐step procedure. The diethylenetriaminepentaacetic acid/Eu³⁺‐labeled R3/I5 bound both receptors RXFP3 and RXFP4 with high binding affinities and low nonspecific binding. Thus, we have presented a valuable nonradioactive tracer for future interaction studies on RXFP3 and RXFP4 with various natural or designed ligands. The present approach could also be adapted for preparing and labeling of other chimeric relaxin family peptides. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

Background  

Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors.     

Cdh13 and AdipoQ gene knockout alter instrumental and Pavlovian drug conditioning

Genome‐wide association studies in humans have suggested that variants of the cadherin‐13 (CDH13) gene are associated with substance use disorder, subjective response to amphetamine, and attention deficit hyperactivity disorder. To examine the role of the Cdh13 and its peptide ligand adiponectin (AdipoQ) in addiction‐related behaviors, we assessed Cdh13 knockout (KO) rats and AdipoQ KO mice using intravenous cocaine self‐administration and conditioned place preference (CPP) paradigms. During intravenous cocaine self‐administration, male Cdh13 heterozygous (+/?) and KO (?/?) rats showed increased cue‐induced reinstatement compared with wild‐type (WT) rats when presented with a cocaine‐paired stimulus, whereas female Cdh13 rats showed no differences across genotype. Cdh13 ?/? rats showed higher responding for a saccharin reinforcer and learned the choice reaction time (RT) task more slowly than WTs. However, we found no differences between Cdh13 ?/? and +/+ rats in responding for sensory reinforcement, number of premature responses in the RT task, tendency to approach a Pavlovian food cue, CPP and locomotor activation to cocaine (10 or 20 mg/kg). In AdipoQ ?/? mice, there was a significant increase in CPP to methamphetamine (1 mg/kg) but not to a range of d‐amphetamine doses (0.5, 1, 2 and 4 mg/kg). Taken together, these data suggest that Cdh13 and AdipoQ regulate sensitivity to psychomotor stimulants and palatable rewards without producing major changes in other behaviors. In humans, these two genes may regulate sensitivity to natural and drug rewards, thus influencing susceptibility to the conditioned drug effects and relapse.     

Inhibition of NAALADase by 2‐PMPA attenuates cocaine‐induced relapse in rats: a NAAG‐mGluR2/3‐mediated mechanism

Pharmacological activation of group II metabotropic glutamate receptors (mGluR2/3) inhibits cocaine self‐administration and reinstatement of drug‐seeking behavior, suggesting a possible use of mGluR2/3 agonists in the treatment of cocaine dependence. In this study, we investigated whether elevation of the endogenous mGluR2/3 ligand N‐acetyl‐aspartatylglutamate (NAAG) levels by the N‐acetylated‐alpha‐linked‐acidic dipeptidase inhibitor 2‐(phosphonomethyl)pentanedioic acid (2‐PMPA) attenuates cocaine self‐administration and cocaine‐induced reinstatement of drug seeking. N‐acetylated‐alpha‐linked‐acidic dipeptidase is a NAAG degradation enzyme that hydrolyzes NAAG to N‐acetylaspartate and glutamate. Systemic administration of 2‐PMPA (10‐100 mg/kg, i.p.) inhibited intravenous self‐administration maintained by low unit doses of cocaine and cocaine (but not sucrose)‐induced reinstatement of drug‐seeking behavior. Microinjections of 2‐PMPA (3–5 μg/side) or NAAG (3–5 μg/side) into the nucleus accumbens (NAc), but not into the dorsal striatum, also inhibited cocaine‐induced reinstatement, an effect that was blocked by intra‐NAc injection of LY341495, a selective mGluR2/3 antagonist. In vivo microdialysis demonstrated that 2‐PMPA (10‐100 mg/kg, i.p.) produced a dose‐dependent reduction in both extracellular dopamine (DA) and glutamate, an effect that was also blocked by LY341495. Finally, pre‐treatment with 2‐PMPA partially attenuated cocaine‐enhanced extracellular NAc DA, while completely blocking cocaine‐enhanced extracellular NAc glutamate in rats during reinstatement testing. Intra‐NAc perfusion of LY341495 blocked 2‐PMPA‐induced reductions in cocaine‐enhanced extracellular NAc glutamate, but not DA. These findings suggest that 2‐PMPA is effective in attenuating cocaine‐induced reinstatement of drug‐seeking behavior, likely by attenuating cocaine‐induced increases in NAc DA and glutamate via pre‐synaptic mGluR2/3s.     

Sensitivity to Chronic Methamphetamine Administration and Withdrawal in Mice with Relaxin-3/RXFP3 Deficiency

Methamphetamine (METH) is a highly addictive psychostimulant, and cessation of use is associated with reduced monoamine signalling, and increased anxiety/depressive states. Neurons expressing the neuropeptide, relaxin-3 (RLN3), and its cognate receptor, RXFP3, constitute a putative ‘ascending arousal system’, which shares neuroanatomical and functional similarities with serotonin (5-HT)/dorsal raphe and noradrenaline (NA)/locus coeruleus monoamine systems. In light of possible synergistic roles of RLN3 and 5-HT/NA, endogenous RLN3/RXFP3 signalling may compensate for the temporary reduction in monoamine signalling associated with chronic METH withdrawal, which could alter the profile of ‘behavioural despair’, bodyweight reductions, and increases in anhedonia and anxiety-like behaviours observed following chronic METH administration. In studies to test this theory, Rln3 and Rxfp3 knockout (KO) mice and their wildtype (WT) littermates were injected once daily with saline or escalating doses of METH (2 mg/kg, i.p. on day 1, 4 mg/kg, i.p. on day 2 and 6 mg/kg, i.p. on day 3–10). WT and Rln3 and Rxfp3 KO mice displayed an equivalent sensitivity to behavioural despair (Porsolt swim) during the 2-day METH withdrawal and similar bodyweight reductions on day 3 of METH treatment. Furthermore, during a 3-week period after the cessation of chronic METH exposure, Rln3 KO, Rxfp3 KO and corresponding WT mice displayed similar behavioural responses in paradigms that measured anxiety (light/dark box, elevated plus maze), anhedonia (saccharin preference), and social interaction. These findings indicate that a whole-of-life deficiency in endogenous RLN3/RXFP3 signalling does not markedly alter behavioural sensitivity to chronic METH treatment or withdrawal, but leave open the possibility of a more significant interaction with global or localised manipulations of this peptide system in the adult brain.     

Sex differences in the effects of chronic stress and food restriction on body weight gain and brain expression of CRF and relaxin‐3 in rats

This study investigated sex‐specific effects of repeated stress and food restriction on food intake, body weight, corticosterone plasma levels and expression of corticotropin‐releasing factor (CRF) in the hypothalamus and relaxin‐3 in the nucleus incertus (NI). The CRF and relaxin‐3 expression is affected by stress, and these neuropeptides produce opposite effects on feeding (anorexigenic and orexigenic, respectively), but sex‐specific regulation of CRF and relaxin‐3 by chronic stress is not fully understood. Male and female rats were fed ad libitum chow (AC) or ad libitum chow and intermittent palatable liquid Ensure without food restriction (ACE), or combined with repeated food restriction (60% chow, 2 days per week; RCE). Half of the rats were submitted to 1‐h restraint stress once a week. In total, seven weekly cycles were applied. The body weight of the RCE stressed male rats significantly decreased, whereas the body weight of the RCE stressed female rats significantly increased compared with the respective control groups. The stressed female RCE rats considerably overate chow during recovery from stress and food restriction. The RCE female rats showed elevated plasma corticosterone levels and low expression of CRF mRNA in the paraventricular hypothalamic nucleus but not in the medial preoptic area. The NI expression of relaxin‐3 mRNA was significantly higher in the stressed RCE female rats compared with other groups. An increase in the expression of orexigenic relaxin‐3 and misbalanced hypothalamic‐pituitary‐adrenal axis activity may contribute to the overeating and increased body weight seen in chronically stressed and repeatedly food‐restricted female rats .     

Sub‐picomolar relaxin signalling by a pre‐assembled RXFP1, AKAP79, AC2, β‐arrestin 2, PDE4D3 complex

Biochemical studies suggest that G‐protein‐coupled receptors (GPCRs) achieve exquisite signalling specificity by forming selective complexes, termed signalosomes. Here, using cAMP biosensors in single cells, we uncover a pre‐assembled, constitutively active GPCR signalosome, that couples the relaxin receptor, relaxin family peptide receptor 1 (RXFP1), to cAMP following receptor stimulation with sub‐picomolar concentrations of peptide. The physiological effects of relaxin, a pleiotropic hormone with therapeutic potential in cancer metastasis and heart failure, are generally attributed to local production of the peptide, that occur in response to sub‐micromolar concentrations. The highly sensitive signalosome identified here provides a regulatory mechanism for the extremely low levels of relaxin that circulate. The signalosome includes requisite Gα_s, Gβγ and adenylyl cyclase 2 (AC2); AC2 is functionally coupled to RXFP1 through AKAP79 binding to helix 8 of the receptor; activation of AC2 is tonically opposed by protein kinase A (PKA)‐activated PDE4D3, scaffolded through a β‐arrestin 2 interaction with Ser⁷⁰⁴ of the receptor C‐terminus. This elaborate, pre‐assembled, ligand‐independent GPCR signalosome represents a new paradigm in GPCR signalling and provides a mechanism for the distal actions of low circulating levels of relaxin.     

Zfp462 deficiency causes anxiety‐like behaviors with excessive self‐grooming in mice

Zfp462 is a newly identified vertebrate‐specific zinc finger protein that contains nearly 2500 amino acids and 23 putative C2H2‐type zinc finger domains. So far, the functions of Zfp462 remain unclear. In our study, we showed that Zfp462 is expressed predominantly in the developing brain, especially in the cerebral cortex and hippocampus regions from embryonic day 7.5 to early postnatal stage. By using a piggyBac transposon‐generated Zfp462 knockout (KO) mouse model, we found that Zfp462 KO mice exhibited prenatal lethality with normal neural tube patterning, whereas heterozygous (Het) Zfp462 KO (Zfp462^+/?) mice showed developmental delay with low body weight and brain weight. Behavioral studies showed that Zfp462^+/? mice presented anxiety‐like behaviors with excessive self‐grooming and hair loss, which were similar to the pathological grooming behaviors in Hoxb8 KO mice. Further analysis of grooming microstructure showed the impairment of grooming patterning in Zfp462^+/? mice. In addition, the mRNA levels of Pbx1 (pre‐B‐cell leukemia homeobox 1, an interacting protein of Zfp462) and Hoxb8 decreased in the brains of Zfp462^+/? mice, which may be the cause of anxiety‐like behaviors. Finally, imipramine, a widely used and effective anti‐anxiety medicine, rescued anxiety‐like behaviors and excessive self‐grooming in Zfp462^+/? mice. In conclusion, Zfp462 deficiency causes anxiety‐like behaviors with excessive self‐grooming in mice. This provides a novel genetic mouse model for anxiety disorders and a useful tool to determine potential therapeutic targets for anxiety disorders and screen anti‐anxiety drugs.     

Regional registration of [6‐14C]glucose metabolism during brain activation of α‐syntrophin knockout mice

α‐Syntrophin is a component of the dystrophin scaffold‐protein complex that serves as an adaptor for recruitment of key proteins to the cytoplasmic side of plasma membranes. α‐Syntrophin knockout (KO) causes loss of the polarized localization of aquaporin4 (AQP4) at astrocytic endfeet and interferes with water and K⁺ homeostasis. During brain activation, release of ions and metabolites from endfeet is anticipated to increase perivascular fluid osmolarity, AQP4‐mediated osmotic water flow from endfeet, and metabolite washout from brain. This study tests the hypothesis that reduced levels of endfoot AQP4 increase retention of [¹⁴C]metabolites during sensory stimulation. Conscious KO and wild‐type mice were pulse‐labeled with [6‐¹⁴C] glucose during unilateral acoustic stimulation or bilateral acoustic plus whisker stimulation, and label retention was assayed by computer‐assisted brain imaging or analysis of [¹⁴C]metabolites in extracts, respectively. High‐resolution autoradiographic assays detected a 17% side‐to‐side difference (p < 0.05) in inferior colliculus of KO mice, not wild‐type mice. However, there were no labeling differences between KO and wild‐type mice for five major HPLC fractions from four dissected regions, presumably because of insufficient anatomical resolution. The results suggest a role for AQP4‐mediated water flow in support of washout of metabolites, and underscore the need for greater understanding of astrocytic water and metabolite fluxes.     

A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism

Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 ^DG/DG mice compared to Rxfp2 ^+/DG and Rxfp2 ^+/+ mice. Testicular histology of the Rxfp2 ^DG/DG mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 ^?) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 ^DG/?) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 ^+/? littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7–9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.     

Unique gene alterations are induced in FACS‐purified Fos‐positive neurons activated during cue‐induced relapse to heroin seeking

Cue‐induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non‐activated neurons during cue‐induced heroin seeking after prolonged withdrawal. We trained rats to self‐administer heroin for 10 days (6 h/day) and assessed cue‐induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent‐activated cell sorting (FACS) to purify Fos‐positive and Fos‐negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos‐immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos‐positive, but not Fos‐negative, neurons. In support of these findings, double‐label immunohistochemistry indicated substantial coexpression of neuropeptide Y (NPY)‐ and Arc‐immunoreactivity in Fos‐positive neurons. Our data indicate that cue‐induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non‐activated neurons.     

Discovery of a small molecule RXFP3/4 agonist that increases food intake in rats upon acute central administration

The relaxin family peptide receptors have been implicated in numerous physiological processes including energy homeostasis, cardiac function, wound healing, and reproductive function. Two family members, RXFP3 and RXFP4, are class A GPCRs with endogenous peptide ligands (relaxin-3 and insulin-like peptide 5 (INSL5), respectively). Polymorphisms in relaxin-3 and RXFP3 have been associated with obesity, diabetes, and hypercholesterolemia. Moreover, central administration of relaxin-3 in rats has been shown to increase food intake, leading to body weight gain. Reported RXFP3 and RXFP4 ligands have been restricted to peptides (both endogenous and synthetic) as well as a low molecular weight positive allosteric modulator requiring a non-endogenous orthosteric ligand. Described here is the discovery of the first potent low molecular weight dual agonists of RXFP3/4. The scaffold identified is competitive with a chimeric relaxin-3/INSL5 peptide for RXFP3 binding, elicits similar downstream signaling as relaxin-3, and increases food intake in rats following acute central administration. This is the first report of small molecule RXFP3/4 agonism.     

Phosphodiesterase‐1b deletion confers depression‐like behavioral resistance separate from stress‐related effects in mice

Phosphodiesterase‐1b (Pde1b) is highly expressed in striatum, dentate gyrus, CA3 and substantia nigra. In a new Floxed Pde1b × Cre^CMV global knockout (KO) mouse model, we show an immobility‐resistance phenotype that recapitulates that found in constitutive Pde1b KO mice. We use this new mouse model to show that the resistance to acute stress‐induced depression‐like phenotype is not the product of changes in locomotor activity or reactivity to other stressors (learned helplessness, novelty suppressed feeding or dexamethasone suppression), and is not associated with anhedonia using the sucrose preference test. Using tamoxifen inducible Cre, we show that the immobility‐resistant phenotype depends on the age of induction. The effect is present when Pde1b is Reduced from conception, P0 or P32, but not if reduced as adults (P60). We also mapped regional brain expression of PDE1B protein and of the Cre driver. These data add to the suggestion that PDE1B may be a target for drug development with therapeutic potential in depression alone or in combination with existing antidepressants.     

Obesity in Insulin Receptor Substrate‐2–Deficient Mice: Disrupted Control of Arcuate Nucleus Neuropeptides

Objectives : Disturbances in insulin signaling have been shown to induce obesity and/or hyperphagia in brain insulin receptor or insulin receptor substrate‐2 (IRS‐2) knockout (KO) mice. This study aimed to examine the central and peripheral mechanisms underlying the phenotype in IRS‐2 KO mice. Research Methods and Procedures : We measured the histological characterization of adipose tissues, mRNA levels of pro‐opiomelanocortin, agouti‐related protein, and neuropeptide Y in the hypothalamus and uncoupling proteins (UCPs) in peripheral tissues of IRS‐2 KO mice. Results : Female IRS‐2 KO mice showed increased daily food intake. Body weight and adiposity were increased in both sexes, although these differences were more pronounced in female than in male IRS‐2 KO mice. Both male and female IRS‐2 KO mice showed decreased UCP1 mRNA expression in brown adipose tissue with defective thermoregulation, and UCP2 mRNA expression was increased in the white adipose tissue of female knockout mice. Furthermore, arcuate nucleus mRNA expression of pro‐opiomelanocortin, was decreased in both male and female IRS‐2 KO mice, whereas expression of agouti‐related protein and neuropeptide Y were increased in female IRS‐2 KO mice. Discussion : In IRS‐2 KO mice, disrupted control of hypothalamic neuropeptide levels and UCP mRNA expression may contribute to the development of obesity.     

TPH2 5′‐ and 3′‐regulatory polymorphisms are differentially associated with HPA axis function and self‐injurious behavior in rhesus monkeys

Tryptophan hydroxylase‐2 (TPH2) synthesizes neuronal serotonin and is linked to numerous behavioral traits. We have previously characterized the functionality of polymorphisms (especially 2051A>C) in 3’‐untranslated region (3’‐UTR) of rhesus monkey TPH2 (rhTPH2). This study further assessed the functionality of additional polymorphisms (–1605T>C, –1491Tn, –1485(AT)n, –1454A>G, –1325In>Del and –363T>G) in rhTPH2 5’‐flanking region (5’‐FR), and evaluated the effects of rhTPH2 5’ and 3’ genotypes on central serotonin turnover, hypothalamic–pituitary–adrenal (HPA) axis function and self‐injurious behavior (SIB) in 32 unrelated adult male monkeys of Indian origin. Haplotypes of the rhTPH2 5’‐FR polymorphisms exert a significant, cell‐dependent effect on reporter gene expression, primarily conferred by –1485(AT)n. The –1485(AT)n and 2051A>C polymorphisms interact to influence cerebrospinal fluid (CSF) 5‐HIAA and plasma adrenocorticotropic hormone (ACTH) in the afternoon. While –1485(AT)n exerts significant main effects on the afternoon cortisol level and nocturnal HPA negative feedback, 2051A>C has significant main effects on the morning cortisol level and cortisol response to ACTH challenge, as well as marginally significant main effects on the daytime HPA negative feedback and self‐biting rate. In addition, the genotype/allele frequency of the 5’‐FR –1325Ins>Del differed significantly between the self‐wounders and non‐wounders, whereas 3’‐UTR 2128S>L polymorphism differed significantly in genotype/allele frequency between the high‐ and low‐frequency biters. This study shows the functionality of rhTPH2 5’‐FR polymorphisms, and provides evidence for the differential association of rhTPH2 5’‐FR and 3’‐UTR polymorphisms with HPA axis function and SIB. Our findings shed light on the role of TPH2 gene variance in physiology and behavioral traits, and also contribute to the understanding of the pathophysiology and genetics of SIB     

Sirt3 is essential for apelin‐induced angiogenesis in post‐myocardial infarction of diabetes

Heart failure following myocardial infarction (MI) is the leading cause of death in diabetic patients. Angiogenesis contributes to cardiac repair and functional recovery in post‐MI. Our previous study shows that apelin (APLN) increases Sirtuin 3 (Sirt3) expression and ameliorates diabetic cardiomyopathy. In this study, we further investigated the direct role of Sirt3 in APLN‐induced angiogenesis in post‐MI model of diabetes. Wild‐type (WT) and Sirt3 knockout (Sirt3KO) mice were induced into diabetes by i.p. streptozotocin (STZ). STZ mice were then subjected to MI followed by immediate intramyocardial injection with adenovirus‐apelin (Ad‐APLN). Our studies showed that Sirt3 expression was significantly reduced in the hearts of STZ mice. Ad‐APLN treatment resulted in up‐regulation of Sirt3, angiopoietins/Tie‐2 and VEGF/VEGFR2 expression together with increased myocardial vascular densities in WT‐STZ+MI mice, but these alterations were not observed in Sirt3KO‐STZ+MI mice. In vitro, overexpression of APLN increased Sirt3 expression and angiogenesis in endothelial progenitor cells (EPC) from WT mice, but not in EPC from Sirt3KO mice. APLN gene therapy increases angiogenesis and improves cardiac functional recovery in diabetic hearts via up‐regulation of Sirt3 pathway.     

Sirtuin3 deficiency exacerbates carbon tetrachloride−induced hepatic injury in mice

Sirtuin3 (SIRT3) plays an important role in maintaining normal mitochondrial function and alleviating oxidative stress. After carbon tetrachloride (CCl₄) administration, the expression of SIRT3 decreased in the liver of mice, which indicated that the SIRT3 might play a crucial role during chemical‐induced acute hepatic injury. To verify the hypothesis, CCl ₄ was given to induce acute hepatic injury in SIRT3 knockout (KO) mice and wild‐type (WT) mice. CCl ₄‐induced liver injury was more severe in SIRT3 KO mice compared with the WT mice. In addition, the oxidative stress induced by CCl ₄ was enhanced in the SIRT3 KO mice. Furthermore, the increased expression of dynamin‐related protein 1 was also aggravated in SIRT3 KO mice after CCl ₄ administration. In conclusion, our study demonstrated that SIRT3 deficiency exacerbated CCl ₄‐induced impairment of the liver in mice, and the mechanism might be related to enhanced oxidative stress.