beta(1) Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple beta-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the beta(1) isoform in energy homeostasis. First, the 30 min i.v. infusion of norepinephrine (NE) or the beta(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the beta(1) gene (KO of beta(1) adrenergic receptor (beta 1KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, beta 1KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, beta 1KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the beta(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis. Journal of Endocrinology (2012) 214, 359-365

HYDROGEN SULFIDE AS A CRYOGENIC MEDIATOR OF HYPDXIA-INDUCED ANAPYREXIA

Hypoxia causes a regulated decrease in body temperature (Tb), a response that has been aptly called anapyrexia, but the mechanisms involved are not completely understood. The roles played by nitric oxide (NO) and other neurotransmitters have been documented during hypoxia-induced anapyrexia, but no information exists with respect to hydrogen sulfide (H(2)S), a gaseous molecule endogenously produced by cystathionine beta-synthase (CBS). We tested the hypothesis that HA production is enhanced during hypoxia and that the gas acts in the anteroventral preoptic region (AVPO; the most important thermosensitive and thermointegrative region of the CNS) modulating hypoxia-induced anapyrexia. Thus, we assessed CBS and nitric oxide synthase (NOS) activities [by means of H2S and nitrite/nitrate (NO(x)) production, respectively] as well as cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) levels in the anteroventral third ventricle region (AV3V; where the AVPO is located) during normoxia and hypoxia. Furthermore, we evaluated the effects of pharmacological modifiers of the H2S pathway given i.c.v. or intra-AVPO. I.c.v. or intra-AVPO microinjection of CBS inhibitor caused no change in Tb under normoxia but significantly attenuated hypoxia-induced anapyrexia. During hypoxia there were concurrent increases in H2S production, which could be prevented by CBS inhibitor, indicating the endogenous source of the gas. cAMP concentration, but not cGMP and NOR, correlated with CBS activity. CBS inhibition increased NOS activity, whereas H2S donor decreased NO. production. In conclusion, hypoxia activates H2S endogenous production through the CBS-H(2)S pathway in the AVPO, having a cryogenic effect. Moreover, the present data are consistent with the notion that the two gaseous molecules, H(2)S and NO, play a key role in mediating the drop in Tb caused by hypoxia and that a fine-balanced interplay between NOS-NO and CBS-H(2)S pathways takes place in the AVPO of rats exposed to hypoxia. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Uptake And Metabolism Of 5’-AMP In The Erythrocyte Play Key Roles In The 5’-AMP Induced Model Of Deep Hypometabolism

UPTAKE AND METABOLISM OF 5’-AMP IN THE ERYTHROCYTE PLAY KEY ROLES IN THE 5’-AMP INDUCED MODEL OF DEEP HYPOMETABOLISM Publication No. ________ Isadora Susan Daniels, B.A. Supervisory Professor: Cheng Chi Lee, Ph.D. Mechanisms that initiate and control the natural hypometabolic states of mammals are poorly understood. The laboratory developed a model of deep hypometabolism (DH) initiated by uptake of 5’-adenosine monophosphate (5’-AMP) into erythrocytes. Mice enter DH when given a high dose of 5’-AMP and the body cools readily. Influx of 5’-AMP appears to inhibit thermoregulatory control. In a 15°C environment, mice injected with 5’-AMP (0.5 mg/gw) enter a Phase I response in which oxygen consumption (VO2) drops rapidly to 1/3rd of euthermic levels. The Phase I response appears independent of body temperature (Tb). This is followed by gradual body temperature decline that correlates with VO2 decline, called Phase II response. Within 90 minutes, mouse Tb approaches 15°C, and VO2 is 1/10th of normal. Mice can remain several hours in this state, before gradually and safely recovering. The DH state translates to other mammalian species. Our studies show uptake and metabolism of 5’-AMP in erythrocytes causes biochemical changes that initiate DH. Increased AMP shifts the adenylate equilibrium toward ADP formation, consequently decreasing intracellular ATP. In turn, glycolysis slows, indicated by increased glucose and decreased lactate. 2,3-bisphosphoglycerate levels rise, allosterically reducing oxygen affinity for hemoglobin, and deoxyhemoglobin rises. Less oxygen transport to tissues likely triggers the DH model. The major intracellular pathway for AMP catabolism is catalyzed by AMP deaminase (AMPD). Multiple AMPD isozymes are expressed in various tissues, but erythrocytes only have AMPD3. Mice lacking AMPD3 were created to study control of the DH model, specifically in erythrocytes. Telemetric measurements demonstrate lower Tb and difficulty maintaining Tb under moderate metabolic stress. A more dramatic response to lower dose of 5’-AMP suggests AMPD activity in the erythrocyte plays an important role in control of the DH model. Analysis of adenylates in erythrocyte lysate shows 3-fold higher levels of ATP and ADP but similar AMP levels to wild-type. Taken together, results indicate alterations in energy status of erythrocytes can induce a hypometabolic state. AMPD3 control of AMP catabolism is important in controlling the DH model. Genetically reducing AMP catabolism in erythrocytes causes a phenotype of lower Tb and compromised ability to maintain temperature homeostasis.

Torpor in mice is induced by both leptin-dependent and -independent mechanisms

We tested the effect of chronic leptin treatment on fasting-induced torpor in leptin-deficient A-ZIP/F-1 and ob/ob mice. A-ZIP/F-1 mice have virtually no white adipose tissue and low leptin levels, whereas ob/ob mice have an abundance of fat but no leptin. These two models allowed us to examine the roles of adipose tissue and leptin in the regulation of entry into torpor. Torpor is a short-term hibernation-like state that allows conservation of metabolic fuels. We first characterized the A-ZIP/F-1 animals, which have a 10-fold reduction in total body triglyceride stores. Upon fasting, A-ZIP/F-1 mice develop a lower metabolic rate and decreased plasma glucose, insulin, and triglyceride levels, with no increase in free fatty acids or β-hydroxybutyrate. Unlike control mice, by 24 hr of fasting, they have nearly exhausted their triglycerides and are catabolizing protein. To conserve energy supplies during fasting, A-ZIP/F-1 (but not control) mice entered deep torpor, with a minimum core body temperature of 24°C, 2°C above ambient. In ob/ob mice, fasting-induced torpor was completely reversed by leptin treatment. In contrast, neither leptin nor thyroid hormone prevented torpor in A-ZIP/F-1 mice. These data suggest that there are at least two signals for entry into torpor in mice, a low leptin level and another signal that is independent of leptin and thyroid hormone levels. Studying rodent torpor provides insight into human torpor-like states such as near drowning in cold water and induced hypothermia for surgery.

Molecular changes induced by a centrally-induced state of synthetic torpor in multiple organs:a new strategy for radioprotection.

Torpor is a successful survival strategy displayed by several mammalian species to cope with harsh environmental conditions. A complex interplay of ambient, genetic and circadian stimuli acts centrally to induce a severe suppression of metabolic rate, usually followed by an apparently undefended reduction of body temperature. Some animals, such as marmots, are able to maintain this physiological state for months (hibernation), during which torpor bouts are periodically interrupted by short interbouts of normothermia (arousals). Interestingly, torpor adaptations have been shown to be associated with a large resistance towards stressors, such as radiation: indeed, if irradiated during torpor, hibernators can tolerate higher doses of radiation, showing an increased survival rate. New insights for radiotherapy and long-term space exploration could arise from the induction of torpor in non-hibernators, like humans. The present research project is centered on synthetic torpor (ST), a hypometabolic/hypothermic condition induced in a non-hibernator, the rat, through the pharmacological inhibition of the Raphe Pallidus, a key brainstem area controlling thermogenic effectors. By exploiting this procedure, this thesis aimed at: i) providing a multiorgan description of the functional cellular adaptations to ST; ii) exploring the possibility, and the underpinning molecular mechanisms, of enhanced radioresistance induced by ST. To achieve these aims, transcriptional and histological analysis have been performed in multiple organs of synthetic torpid rats and normothermic rats, either exposed or not exposed to 3 Gy total body of X-rays. The results showed that: i) similarly to natural torpor, ST induction leads to the activation of survival and stress resistance responses, which allow the organs to successfully adapt to the new homeostasis; ii) ST provides tissue protection against radiation damage, probably mainly through the cellular adaptations constitutively induced by ST, even though the triggering of specific responses when the animal is irradiated during hypothermia might play a role.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

Low-density Lipoprotein Cholesterol And Radiotherapy-induced Carotid Atherosclerosis In Subjects With Head And Neck Cancer.

Radiotherapy (RT) is a risk factor for accelerated carotid artery atherosclerotic disease in subjects with head and neck cancer. However, the risk factors of RT-induced carotid artery remodeling are not established. This study aimed to investigate the effects of RT on carotid and popliteal arteries in subjects with head and neck cancer and to evaluate the relationship between baseline clinical and laboratory features and the progression of RT-induced atherosclerosis. Eleven men (age = 57.9 ± 6.2years) with head and neck cancer who underwent cervical bilateral irradiation were prospectively examined by clinical and laboratory analysis and by carotid and popliteal ultrasound before and after treatment (mean interval between the end of RT and the post-RT assessment = 181 ± 47 days). No studied subject used hypocholesterolemic medications. Significant increases in carotid intima-media thickness (IMT) (0.95 ± 0.08 vs. 0.87 ± 0.05 mm; p < 0.0001) and carotid IMT/diameter ratio (0.138 ± 0.013 vs. 0.129 ± 0.014; p = 0.001) were observed after RT, while no changes in popliteal structural features were detected. In addition, baseline low-density lipoprotein cholesterol levels showed a direct correlation with RT-induced carotid IMT change (r = 0.66; p = 0.027), while no other studied variable exhibited a significant relationship with carotid IMT change. These results indicate that RT-induced atherosclerosis is limited to the irradiated area and also suggest that it may be predicted by low-density lipoprotein cholesterol levels in subjects with head and neck cancer.

Peripheral P2x7 Receptor-induced Mechanical Hyperalgesia Is Mediated By Bradykinin.

P2X7 receptors play an important role in inflammatory hyperalgesia, but the mechanisms involved in their hyperalgesic role are not completely understood. In this study, we hypothesized that P2X7 receptor activation induces mechanical hyperalgesia via the inflammatory mediators bradykinin, sympathomimetic amines, prostaglandin E2 (PGE2), and pro-inflammatory cytokines and via neutrophil migration in rats. We found that 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP), the most potent P2X7 receptor agonist available, induced a dose-dependent mechanical hyperalgesia that was blocked by the P2X7 receptor-selective antagonist A-438079 but unaffected by the P2X1,3,2/3 receptor antagonist TNP-ATP. These findings confirm that, although BzATP also acts at both P2X1 and P2X3 receptors, BzATP-induced hyperalgesia was mediated only by P2X7 receptor activation. Co-administration of selective antagonists of bradykinin B1 (Des-Arg(8)-Leu(9)-BK (DALBK)) or B2 receptors (bradyzide), β1 (atenolol) or β2 adrenoceptors (ICI 118,551), or local pre-treatment with the cyclooxygenase inhibitor indomethacin or the nonspecific selectin inhibitor fucoidan each significantly reduced BzATP-induced mechanical hyperalgesia in the rat hind paw. BzATP also induced the release of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), an effect that was significantly reduced by A-438079. Co-administration of DALBK or bradyzide with BzATP significantly reduced BzATP-induced IL-1β and CINC-1 release. These results indicate that peripheral P2X7 receptor activation induces mechanical hyperalgesia via inflammatory mediators, especially bradykinin, which may contribute to pro-inflammatory cytokine release. These pro-inflammatory cytokines in turn may mediate the contributions of PGE2, sympathomimetic amines and neutrophil migration to the mechanical hyperalgesia induced by local P2X7 receptor activation.

15-deoxy-Δ12,14-prostaglandin J2 Reduces Albumin-induced Arthritis In Temporomandibular Joint Of Rats.

The aim of this study was to evaluate the peripheral effect of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in albumin-induced arthritis in temporomandibular joint (TMJ) of rats. Antigen-induced arthritis (AIA) was generated in rats with methylated bovine serum albumin (mBSA) diluted in complete Freund׳s adjuvant. Pretreatment with an intra-articular injection of 15d-PGJ2 (100 ng/TMJ) before mBSA intra-articular injection (10 µg/TMJ) (challenge) in immunized rats significantly reduced the albumin-induced arthritis inflammation. The results demonstrated that 15d-PGJ2 was able to inhibit plasma extravasation, leukocyte migration and the release of inflammatory cytokines IL-6, IL-12, IL-18 and the chemokine CINC-1 in the TMJ tissues. In addition, 15d-PGJ2 was able to increase the expression of the anti-adhesive molecule CD55 and the anti-inflammatory cytokine IL-10. Taken together, it is possible to suggest that 15d-PGJ2 inhibit leukocyte infiltration and subsequently inflammatory process, through a shift in the balance of the pro- and anti-adhesive properties. Thus, 15d-PGJ2 might be used as a potential anti-inflammatory drug to treat arthritis-induced inflammation of the temporomandibular joint.

Hypothermia And Early Neonatal Mortality In Preterm Infants.

To evaluate intervention practices associated with hypothermia at both 5 minutes after birth and at neonatal intensive care unit (NICU) admission and to determine whether hypothermia at NICU admission is associated with early neonatal death in preterm infants. This prospective cohort included 1764 inborn neonates of 22-33 weeks without malformations admitted to 9 university NICUs from August 2010 through April 2012. All centers followed neonatal International Liaison Committee on Resuscitation recommendations for the stabilization and resuscitation in the delivery room (DR). Variables associated with hypothermia (axillary temperature <36.0 °C) 5 minutes after birth and at NICU admission, as well as those associated with early death, were analyzed by logistic regression. Hypothermia 5 minutes after birth and at NICU admission was noted in 44% and 51%, respectively, with 6% of early neonatal deaths. Adjusted for confounding variables, practices associated with hypothermia at 5 minutes after birth were DR temperature <25 °C (OR 2.13, 95% CI 1.67-2.28), maternal temperature at delivery <36.0 °C (OR 1.93, 95% CI 1.49-2.51), and use of plastic bag/wrap (OR 0.53, 95% CI 0.40-0.70). The variables associated with hypothermia at NICU admission were DR temperature <25 °C (OR 1.44, 95% CI 1.10-1.88), respiratory support with cold air in the DR (OR 1.40, 95% CI 1.03-1.88) and during transport to NICU (OR 1.51, 95% CI 1.08-2.13), and cap use (OR 0.55, 95% CI 0.39-0.78). Hypothermia at NICU admission increased the chance of early neonatal death by 1.64-fold (95% CI 1.03-2.61). Simple interventions, such as maintaining DR temperature >25 °C, reducing maternal hypothermia prior to delivery, providing plastic bags/wraps and caps for the newly born infants, and using warm resuscitation gases, may decrease hypothermia at NICU admission and improve early neonatal survival.

Uv-vis Spectra As An Alternative To The Lowry Method For Quantify Hair Damage Induced By Surfactants.

It is well known that long term use of shampoo causes damage to human hair. Although the Lowry method has been widely used to quantify hair damage, it is unsuitable to determine this in the presence of some surfactants and there is no other method proposed in literature. In this work, a different method is used to investigate and compare the hair damage induced by four types of surfactants (including three commercial-grade surfactants) and water. Hair samples were immersed in aqueous solution of surfactants under conditions that resemble a shower (38 °C, constant shaking). These solutions become colored with time of contact with hair and its UV-vis spectra were recorded. For comparison, the amount of extracted proteins from hair by sodium dodecyl sulfate (SDS) and by water were estimated by the Lowry method. Additionally, non-pigmented vs. pigmented hair and also sepia melanin were used to understand the washing solution color and their spectra. The results presented herein show that hair degradation is mostly caused by the extraction of proteins, cuticle fragments and melanin granules from hair fiber. It was found that the intensity of solution color varies with the charge density of the surfactants. Furthermore, the intensity of solution color can be correlated to the amount of proteins quantified by the Lowry method as well as to the degree of hair damage. UV-vis spectrum of hair washing solutions is a simple and straightforward method to quantify and compare hair damages induced by different commercial surfactants.

Inactivation Of Ampk Mediates High Phosphate-induced Extracellular Matrix Accumulation Via Nox4/tgfß-1 Signaling In Human Mesangial Cells.

High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs). iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively. In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression. AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.

Tadalafil-induced Improvement In Left Ventricular Diastolic Function In Resistant Hypertension.

Left ventricular hypertrophy and diastolic dysfunction (LVDD) remain highly frequent markers of cardiac damage and risk of progression to symptomatic heart failure, especially in resistant hypertension (RHTN). We have previously demonstrated that administration of sildenafil in hypertensive rats improves LVDD, restoring phosphodiesterase type 5 (PDE-5) inhibition in cardiac myocytes. We hypothesized that the long-acting PDE-5 inhibitor tadalafil may be clinically useful in improving LVDD in RHTN independently of blood pressure (BP) reduction. A single blinded, placebo-controlled, crossover study enrolled 19 patients with both RHTN and LVDD. Firstly, subjects received tadalafil (20 mg) for 14 days and after a 2-week washout period, they received placebo orally for 14 days. Patients were evaluated by office BP and ambulatory BP monitoring (ABPM), endothelial function (FMD), echocardiography, plasma brain natriuretic peptide (BNP-32), cyclic guanosine monophosphate (cGMP) and nitrite levels. No significant differences were detected in BP measurements. Remarkably, at least four echocardiographic parameters related with diastolic function improved accompanied by decrease in BNP-32 in tadalafil use. Although increasing cGMP, tadalafil did not change endothelial function or nitrites. There were no changes in those parameters after placebo. The current findings suggest that tadalafil improves LV relaxation through direct effects PDE-5-mediated in the cardiomyocytes with potential benefit as an adjunct to treat symptomatic subjects with LVDD such as RHTN patients.

Decreased Expression Of Stem Cell Markers By Simvastatin In 7,12-dimethylbenz(a)anthracene (dmba)-induced Breast Cancer.

Simvastatin, a competitive inhibitor of HMG-CoA reductase widely used in the treatment and prevention of hyperlipidemia-related diseases, has recently been associated to in vitro anticancer stem cell (CSC) actions. However, these effects have not been confirmed in vivo. To assess in vivo anti-CSC effects of simvastatin, female Sprague-Dawley rats with 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary cancer and control animals were treated for 14 days with either simvastatin (20 or 40 mg/kg/day) or soybean oil (N = 60). Tumors and normal breast tissues were removed for pathologic examination and immunodetection of CSC markers. At 40 mg/kg/day, simvastatin significantly reduced tumor growth and the expression of most CSC markers. The reduction in tumor growth (80%) could not be explained solely by the decrease in CSCs, since the latter accounted for less than 10% of the neoplasia (differentiated cancer cells were also affected). Stem cells in normal, nonneoplastic breast tissues were not affected by simvastatin. Simvastatin was also associated with a significant decrease in proliferative activity but no increase in cell death. In conclusion, this is the first study to confirm simvastatin anti-CSC actions in vivo, further demonstrating that this effect is specific for neoplastic cells, but not restricted to CSCs, and most likely due to inhibition of cell proliferation.