CL-316,243 Research Articles

Lactate utilization in Lace1 knockout mice promotes browning of inguinal white adipose tissue

Recent studies have focused on identifying novel genes involved in the browning process of inguinal white adipose tissue (iWAT). In this context, we propose that the mitochondrial ATPase gene lactation elevated 1 (Lace1) utilizes lactate to regulate the browning capacity of iWAT, specifically in response to challenge with CL-316,243 (CL), a beta3-adrenergic receptor (β3-AR) agonist. The mice were injected with CL over a span of 3 days and exposed to cold temperatures (4–6 °C) for 1 week. The results revealed a significant increase in Lace1 expression levels during beige adipogenesis. Additionally, a strong positive correlation was observed between Lace1 and Ucp1 mRNA expression in iWAT under browning stimulation. To further explore this phenomenon, we subjected engineered Lace1 KO mice to CL and cold challenges to validate their browning potential. Surprisingly, Lace1 KO mice presented increased oxygen consumption and heat generation upon CL challenge and cold exposure, along with increased expression of genes related to brown adipogenesis. Notably, deletion of Lace1 led to increased lactate uptake and browning in iWAT under CL challenge compared with those of the controls. These unique phenomena stem from increased lactate release due to the inactivation of pyruvate dehydrogenase (PDH) in the hearts of Lace1 KO mice.

Repeatability of brown adipose tissue activation measured by [18F]FDG PET after beta3-adrenergic stimuli in a mouse model

This study aimed to evaluate the repeatability of brown adipose tissue (BAT) activation measured by [18F]FDG-PET after beta3-adrenergic stimuli with CL316243 in mice. MethodsMale C57BL/6 mice underwent [18F]FDG-PET at baseline without stimulation (T0-NS), on three consecutive days after intravenous administration of the selective β3-adrenergic agonist CL316243 (T1-CL, T2-CL, T3-CL), and without stimuli after 1 and 2 weeks (T7-NS and T14-NS). The standardized uptake value (SUVmax), BAT metabolic volume (BMV), and total BAT glycolysis (TBG) were measured in each scanning session, with statistical groupwise comparisons by ANOVA and post hoc Tukey test. ResultsSUVmax, BMV, and TBG values showed no significant differences between the three PET scans without stimuli, but were significantly higher after CL316243 administration (p < 0.0001). The mean coefficient of variation (CoV) of PET within individuals was 49 % at baseline but only 9 % with pharmacological stimulation. ConclusionsThe study demonstrated that administration of the selective β3-adrenergic receptor agonist CL316243 (CL) in mice leads to consistent metabolic activation of brown adipose tissue (BAT), as measured by [18F]FDG-PET. We also demonstrated metabolic activation by repeated pharmacological challenge, without evidence of hysteresis. Thus, the methods used in the current work should serve for further studies on BAT metabolism in experimental animals, with translational value for clinical research.

Abstract P2084: Adipose Tissues Thermogenesis Provides Cardioprotection Against Diastolic Dysfunction Developed In An Obesity-Induced HFpEF Model

Background: Heart failure with preserved ejection fraction (HFpEF) is a major source of morbidity/ mortality in the United States. A systemic inflammatory state established through underlying comorbidities, such as obesity, is thought to develop cardiac dysfunction in HFpEF. Clinical observations associate adiposity with diastolic dysfunction. Herein we demonstrate that adipose tissue is a direct source of diastolic dysfunction and reveal a cardioprotective role of adipose tissue thermogenesis in obesity related HFpEF. Methods and Results: Mice were treated with a β3-AR agonist, CL-316243 (CL), to induce thermogenesis, followed by HFpEF treatment consisting of HFD and L-NAME. Echocardiography revealed that this protected against HFpEF-induced diastolic dysfunction. Whole heart extracts and AT were then evaluated for the signature of β3-AR signaling. In the heart, no signature of β3-AR signaling was observed. In AT, though, various aspects of β3-AR induced thermogenesis were observed. Histological analysis revelated that CL treatment conferred resistance against obesity-induced whitening of interscapular brown adipose tissue (iBAT). Gene and protein expression changes of thermogenic regulators and lipid metabolism enzymes suggested greater thermogenic activity in CL-treated mice. Accordingly, indirect calorimetry showed enhanced energy expenditure, while regression analysis revealed that AT uniquely contributed to this. Transplantation experiments involving CL-treated donor AT and wild-type donors followed by echocardiographic measurements confirmed that the effects of CL were mediated through AT. This approach was complemented with a genetic model we previously used ( Ucp1 -Cre ERT2 ; Cdkn2a fl/fl ) to promote the expansion of beige AT. Here, beige AT expansion protected against diastolic dysfunction and cardiomyocyte hypertrophy developed during HFpEF. Conclusion: Our study indicates enhanced energy expenditure in AT is cardioprotective in obesity related HFpEF. Furthermore, identifying obese AT as a direct source of diastolic dysfunction highlights the therapeutic potential of targeting AT metabolism as a relevant source of diastolic dysfunction.

1674-P: Increased Glucose Uptake in Brown Fat Promotes Energy Expenditure

Brown and beige fat dissipate energy as heat in response to β3-adrenergic receptor (β3AR) stimulation. Strategies to facilitate this response may be employed to promote weight loss and improve health in patients with metabolic disease. Previously, we discovered that GTPase RalA and its GAP complex RalGAP regulate exocytosis of glucose transporter GLUT4. We demonstrated in vivo, that adipose-tissue specific knockout of RalGAP subunit RalGAPB activates RalA and increases glucose uptake 7 times in brown fat (BAT), leading to improved glucose handling on high fat diet (HFD). Here we seek to test the effect of increased glucose uptake in BAT on energy expenditure (EE), using mice with BAT-specific knockout of RalGAPB (the KO mice). After 8 weeks on 45% HFD, the KO mice and control littermates (Ctr) were injected with β3AR agonist CL-316,243 (CL) or PBS for 8 days. Mice assigned to CL were placed in metabolic cages. Respiratory quotient was increased in the KO mice, indicating increased glucose oxidation (likely by BAT, due to increased glucose uptake). EE at room temperature was elevated in the KO mice. This was compensated by increased food uptake, explaining similar body weights between the KO and Ctr mice. CL injections, as expected, induced spikes in EE. Acute response to CL (arbitrarily defined as EE during 2 hours after CL injection) was consistent between daily CL injections in Ctr mice, and was increased twice in the KO mice compared to Ctr. In Ctr mice, 1st CL injection increased average day and night EE; this response gradually declined with consecutive CL injections. In the KO mice, 1st CL injection elicited similar to Ctr mice increase in average day and night EE; and the response to consecutive CL injections was preserved. As anticipated, CL reduced adiposity in Ctr mice, which was further augmented in the KO mice, consistent with finding on EE. We will further test the role of increased glucose uptake by BAT in EE of the KO mice, and will determine the mechanism of sustained CL responsiveness in the KO mice. Disclosure F.Rezayat: None. J.A.Resnick: None. I.M.Farah: None. Y.Skorobogatko: None. A.Saltiel: Board Member; Elgia Therapeutics. Funding National Institutes of Health (P30DK063491)

Combining a β3 adrenergic receptor agonist with alpha-lipoic acid reduces inflammation in male mice with diet-induced obesity.

Beta-3 adrenergic receptors (β3-AR) stimulate lipolysis and thermogenesis in white and brown adipose tissue (WAT and BAT). Obesity increases oxidative stress and inflammation that attenuate AT β3-AR signaling. The objective of this study was to test the hypothesis that the combination of the β3-AR agonist CL-316,243 (CL) and the antioxidant alpha-lipoic acid (ALA) would lower inflammation in diet-induced obesity (DIO) and improve β3-AR function. A total of 40 DIO mice were separated into four groups: Control (per os and intraperitoneal [IP] vehicle); CL alone (0.01 mg/kg IP daily); ALA alone (250 mg/kg in drinking water); or ALA+CL combination, all for 5 weeks. Food intake was similar in all groups; however, mice receiving ALA+CL showed improved body composition and inflammation as well as lower body weight (+1.7 g Control vs. -2.5 g ALA+CL [-7%]; p < 0.01) and percentage of body fat (-9%, p < 0.001). Systemic and epididymal WAT inflammation was lower with ALA+CL than all other groups, with enhanced recruitment of epididymal WAT anti-inflammatory CD206+ M2 macrophages. β3-AR signaling in WAT was enhanced in the combination-treatment group, with higher mRNA and protein levels of thermogenic uncoupling protein 1 and AT lipases. Chronic treatment with ALA and a β3-AR agonist reduces DIO-induced inflammation. AT immune modulation could be a therapeutic target in patients with obesity.

Dietary Iron Deficiency Modulates Adipocyte Iron Homeostasis, Adaptive Thermogenesis, and Obesity in C57BL/6 Mice

BackgroundAdaptive thermogenesis is an iron-demanding pathway, significantly contributing to whole-body energy expenditure. However, the effects of iron-deficient diets on adaptive thermogenesis and obesity remain unknown. ObjectivesWe aimed to determine the impact of dietary iron deficiency on iron homeostasis in adipocytes, adaptive thermogenic capacity, and metabolic consequences in obesity. MethodsC57BL/6 male mice were assigned to either the iron-adequate (IA, 35 ppm) or the iron-deficient group (ID, 3 ppm) at weaning. Upon 8 wk of age, both IA and ID groups received an isocaloric high-fat diet (45% kcal from fat) for 10 wk, maintaining the same iron content. Mice (n = 8) were used to determine the iron status at the systemic and tissue levels and lipid metabolism and inflammatory signaling in adipose tissue. The same mice were used to evaluate cold tolerance (4°C) for 3 h. For assessing adaptive thermogenesis, mice (n = 5) received an intraperitoneal injection of β3-adrenoceptor agonist CL316243 (CL) for 5 d. ResultsCompared with the IA group, the ID group had nonanemic iron deficiency, lower serum ferritin (42.8%, P < 0.01), and greater weight gain (8.67%, P < 0.05) and insulin resistance (159%, P < 0.01), partly due to reduced AMP-activated protein kinase activation (61.0%, P < 0.05). Upon cold exposure, the ID group maintained a core body temperature 2°C lower than the IA group. The ID group had lower iron content (47.0%, P < 0.01) in the inguinal adipose tissue (iWAT) than the IA group, which was associated with impaired adaptive thermogenesis. In response to CL, ID mice showed decreased heat production (P < 0.01) and defective upregulation of beige adipocyte-specific markers, including uncoupling protein 1 (41.1%, P < 0.001), transferrin receptor 1 (47.5%, P < 0.001), and mitochondrial respiratory chain complexes (P < 0.05) compared with IA mice. ConclusionsDietary iron deficiency deregulates iron balance in the iWAT and impairs adaptive thermogenesis, thereby escalating the diet-induced weight gain in C57BL/6 mice.

The β3 Adrenergic Receptor Agonist CL316243 Ameliorates the Metabolic Abnormalities of High-Fat Diet-Fed Rats by Activating AMPK/PGC-1α Signaling in Skeletal Muscle.

PurposeSkeletal muscle has a major influence on whole-body metabolic homeostasis. In the present study, we aimed to determine the metabolic effects of the β3 adrenergic receptor agonist CL316243 (CL) in the skeletal muscle of high-fat diet-fed rats.MethodsSprague-Dawley rats were randomly allocated to three groups, which were fed a control diet (C) or a high-fat diet (HF), and half of the latter were administered 1 mg/kg CL by gavage once weekly (HF+CL), for 12 weeks. At the end of this period, the serum lipid profile and glucose tolerance of the rats were evaluated. In addition, the phosphorylation and protein and mRNA expression of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, and carnitine palmitoyl transferase (CPT)-1b in skeletal muscle were measured by Western blot analysis and qPCR. The direct effects of CL on the phosphorylation (p-) and expression of AMPK, PGC-1α, and CPT-1b were also evaluated by Western blotting and immunofluorescence in L6 myotubes.ResultsCL administration ameliorated the abnormal lipid profile and glucose tolerance of the high-fat diet-fed rats. In addition, the expression of p-AMPK, PGC-1α, and CPT-1b in the soleus muscle was significantly increased by CL. CL (1 µM) also increased the protein expression of p-AMPK, PGC-1α, and CPT-1b in L6 myotubes. However, the effect of CL on PGC-1α protein expression was blocked by the AMPK antagonist compound C, which suggests that CL increases PGC-1α protein expression via AMPK.ConclusionActivation of the β3 adrenergic receptor in skeletal muscle ameliorates the metabolic abnormalities of high-fat diet-fed rats, at least in part via activation of the AMPK/PGC-1α pathway.

Regulatory T cells promote adipocyte beiging in subcutaneous adipose tissue.

Regulatory T cells (Tregs) play essential roles in obesity and diabetes. Here, we report a role of Tregs in enhancing β3-adrenergic receptor agonist CL316243 (CL)-stimulated thermogenic program in subcutaneous adipose tissue (SAT), but not in visceral fat. CL treatment for 7 days increased SAT adipocyte beiging and thermogenic gene expression in male or female mice. Adoptive transfer of Tregs enhanced this CL activity. Such Treg activity lost in male epididymal white adipose tissue (eWAT) and female gonadal gWAT. Adipocyte culture yielded the same conclusion. Tregs enhanced the expression of CL-induced thermogenic genes in SAT from male and female mice. This activity of Tregs reduced or disappeared in adipocytes from eWAT or gWAT. Both CL and Tregs induced much higher UCP-1 (uncoupling protein-1) expression in SAT from females than that from males. A mechanistic study demonstrated a role of Tregs in suppressing the expression of M1 macrophage markers (Tnfa, Il6, iNos, Ip10) and promoting the expression of M2 macrophage markers (Mrc1, Arg1, Il10) in bone-marrow-derived macrophages or in SAT from male or female mice. In female mice with pre-established obesity, Treg adoptive transfer reduced the gWAT weight in 2 weeks. Together with CL treatment, Treg adoptive transfer reduced the SAT weight and further improved CL-induced glucose metabolism and insulin sensitivity in female obese mice, but did not affect CL-induced body weight loss in male or female obese mice. This study revealed a predominant role of Tregs in female mice in promoting adipocyte beiging and thermogenesis in SAT, in part by slanting M2 macrophage polarization.

Iron-Response Element Binding to Iron Regulatory Protein (IRP) 1 and 2 Are Indispensable for Adipose Tissue Browning

Abstract Objectives Intracellular iron homeostasis is tightly regulated in posttranscriptional levels via iron regulatory proteins (IRPs). IRPs bind to the iron-responsive elements (IREs), leading to either mRNA translation or stability. Our recent study demonstrated that iron metabolism is intimately linked with adipose tissue browning and thermogenic activation. However, the role of IRP/IRE interactions in the adipose tissue is poorly understood. We aim to characterize the IRP/IRE interactions in the adipose tissue in terms of depot-specificity and thermogenic potential. Methods To induce adipocyte browning, mice were administrated with beta-3 adrenoceptor agonist CL316243 (CL) for 5 days, and different depots of adipose tissue of epididymal (eWAT), inguinal (iWAT), brown (BAT), and liver were collected. Iron metabolism and thermogenesis were evaluated. To investigate the IRP/IRE binding, electrophoretic mobility shift assay (EMSA) was performed in the cytosolic using the fluorescence-labeled IRE (IR-IRE). To distinguish the IRE binding with IRP1 and 2, the cytosolic fraction from IRP1 and 2 knockout mice were used as positive controls. Results In a normal temperature, the constitutive IRP/IRE binding was found in the BAT, but not in the eWAT and iWAT. In response to CL treatment, iron content and transferrin receptor levels significantly increased in the WAT. Accordingly, the IRE/IRPs binding significantly increased in the CL-treated iWAT. Genetic deletion of IRP1 or 2 poses a marginal impact on constitutively active BAT development, suggesting IRP1 and 2 plays a compensatory role. Unlikely to BAT, the deletion of either IRP1 or 2 failed to induce WAT browning in the IRP1 and 2 knockout mice with CL stimulation. Consistently, both IRE binding to IRP1 and 2 were manifest in the CL treated iWAT, implicating that IRP1 and 2 plays a separate and synergistic function for WAT browning. Conclusions Our study defined the depot-specific iron regulatory metabolism in the adipose tissue using an innovative EMSA method. We demonstrated that, for the first time in our knowledge, IRE binding to both IRP1 and IRP2 is indispensable for the thermogenic activation of WAT, which is distinct from the iron regulatory mechanism found in the BAT. We propose that iron metabolism in the WAT is a novel determinant for WAT browning and thermogenic energy expenditure. Funding Sources None.

The role of microRNA-23b-5p in regulating brown adipogenesis and thermogenic program.

Enhanced brown adipose tissue (BAT) mass and activity have been demonstrated to promote the expenditure of excess stored energy and reduce prevalence of obesity. Cold is known as a potent stimulator of BAT and activates BAT primarily through the β3-adrenergic-cAMP signaling. Here, we performed RNA-sequencing to identify differential miRNAs in mouse BAT upon cold exposure and a total of 20 miRNAs were validated. With the treatment of CL-316,243 (CL) and forskolin (Fsk) in mouse and human differentiated brown adipocyte cells in vitro, miR-23b-5p, miR-133a-3p, miR-135-5p, miR-491-5p, and miR-150-3p expression decreased and miR-455-5p expression increased. Among these deferentially expressed miRNAs, miR-23b-5p expression was differentially regulated in activated and aging mouse BAT and negatively correlated with Ucp1 expression. Overexpression of miR-23b-5p in the precursor cells from BAT revealed no significant effects on lipid accumulation, but diminished mitochondrial function and decreased expression of BAT specific markers. Though luciferase reporter assays did not confirm the positive association of miR-23b-5p with the 3′UTRs of the predicted target Ern1, miR-23b-5p overexpression may affect brown adipocyte thermogenic capacity mainly through regulating genes expression involving in lipolysis and fatty acid β-oxidation pathways. Our results suggest that miRNAs are involved in cold-mediated BAT thermogenic activation and further acknowledged miR-23b-5p as a negative regulator in controlling thermogenic programs, further providing potential molecular therapeutic targets to increase surplus energy and treat obesity.

Iron Deficiency Anemia (IDA) Promotes Visceral Obesity Due to Defective Adipose Tissue Browning (OR09-07-19)

ObjectivesIron deficiency anemia (IDA) is a common disease related to malnutrition. Epidemiological studies revealed a positive correlation between IDA and obesity, but the underlying mechanism is uncertain. Emerging evidence suggests that adipose browning significantly contributes to weight loss by dissipating extra energy into heat. Adipose tissue browning is concurrent with mitochondrial biogenesis, an iron-demanding process. We aimed to investigate whether iron deficiency downregulat adipose tissue browning. MethodsTo simulate the IDA, C57BL/6 mice were fed a high-fat diet in an iron-deficient (3 ppm) setting for 14 weeks compared with an isocaloric iron-sufficient (50 ppm) diet. Iron status was evaluated by measuring serum levels of ferritin, hemoglobin (Hb) and hematocrit (HCt). Iron content of tissue levels was measured by ICP-Mass spectrometer. For assessment of capability to keep the temperature homeostasis, core body temperature and heat release by the infrared camera were determined upon acute cold exposure (4°C). Adipose browning was induced by administrating β3-adrenoceptor agonist, CL316243 (CL) for 5 days. The significance of iron on adipocyte browning was validated in vitro by silencing of the transferrin receptor (TfR), or by iron chelator (DFO, deferoxamine) treatment during beige adipogenesis. ResultsThe chronic iron-deficiency significantly reduced serum ferritin concentration (P < 0.01) with marginal impact on Hb or HCt. Despite no apparent difference in body weight, fat mass was higher in IDA mice (P < 0.001). Consistently, total iron content in the inguinal fat, where adipose browning occurs, was markedly lower in IDA mice. IDA mice were more susceptible to cold treatment, maintaining the body temperature lower than control and defective in heat release. Upon CL stimulation, IDA mice showed reduced expression of uncoupling protein 1 (UCP1) and Cytc, and beige-like morphology in the inguinal fat. Supporting these in vivo results, inhibition of iron import by depleting TfR or reducing liable iron dampened the beige adipogenesis by attenuating brown-specific markers and mitochondrial biogenesis. ConclusionsIDA is an independent risk factor for visceral obesity by decreasing thermogenic energy expenditure. Funding SourcesNational Institutes of Health Grant 1R21HD094273.

AB321. SPR-48 The effects of myrbetriq on detrusor overactivity associated with suprasacral spinal cord injury (SCI) in rats

Objectiveβ3-adrenoceptor agonists (BARA) represent a novel mechanism of action for direct relaxation of urinary bladder smooth muscle. Myrbetriq (MYR) is an FDA approved BARA developed by Astellas Pharma, and has proven to be very useful for treating overactive bladder. Preliminary data from this laboratory demonstrated a remarkable effect of a rat-specific BARA, CL-316,243 (CL), on the hallmark attributes of neurogenic bladder subsequent to spinal cord injury (SCI). This included an increase in true bladder capacity (TBC), a decrease in the number and amplitude of non-voiding contractions (NVC) and an increased filling compliance (C). The current report reflects a formalized preclinical study of both the rat specific BARA, CL, and MYR, in order to provide preclinical support for utilization of MYR in treating SCI patients with neurogenic detrusor overactivity.MethodsFemale rats (4 weeks post-SCI at T9–10, n=43, 10–11/group) were anesthetized with isoflurane and fitted with femoral vein, ureteral diversion and transvesical catheters. The animals were mounted in Ballman restraint cages to which they had been previously acclimated. Conscious cystometry was performed before and after 3 repeated vehicles (Veh 1–3) and 3 escalating 1/2 log doses of either CL or MYR, and their paired repeated vehicle controls (Veh 4–6) at 30-minute intervals. See figure for testing scheme protocol. Data were analyzed using ANOVA with repeated measures. P<0.05 was considered significant.ResultsBoth CL and MYR significantly increased TBC and NVC count, MYR decreased maximal NVC amplitude, and CL increased C, relative to their respective repeated vehicle controls.ConclusionsThe results of these studies support the use BARA for the treatment of NDO secondary to SCI, as at least one of the drugs tested was able to distinguish themselves from their repeated vehicle control groups as having a positive effect for each of the four measures.Funding Source(s)an IIR grant from Astellas

Metabolic activity of brown, "beige," and white adipose tissues in response to chronic adrenergic stimulation in male mice.

Classical brown adipocytes such as those found in interscapular brown adipose tissue (iBAT) represent energy-burning cells, which have been postulated to play a pivotal role in energy metabolism. Brown adipocytes can also be found in white adipose tissue (WAT) depots [e.g., inguinal WAT (iWAT)] following adrenergic stimulation, and they have been referred to as "beige" adipocytes. Whether the presence of these adipocytes, which gives iWAT a beige appearance, can confer a white depot with some thermogenic activity remains to be seen. In consequence, we designed the present study to investigate the metabolic activity of iBAT, iWAT, and epididymal white depots in mice. Mice were either 1) kept at thermoneutrality (30°C), 2) kept at 30°C and treated daily for 14 days with an adrenergic agonist [CL-316,243 (CL)], or 3) housed at 10°C for 14 days. Metabolic activity was assessed using positron emission tomography imaging with fluoro-[(18)F]deoxyglucose (glucose uptake), fluoro-[(18)F]thiaheptadecanoic acid (fatty acid uptake), and [(11)C]acetate (oxidative activity). In each group, substrate uptakes and oxidative activity were measured in anesthetized mice in response to acute CL. Our results revealed iBAT as a major site of metabolic activity, which exhibited enhanced glucose and nonesterified fatty acid uptakes and oxidative activity in response to chronic cold and CL. On the other hand, beige adipose tissue failed to exhibit appreciable increase in oxidative activity in response to chronic cold and CL. Altogether, our results suggest that the contribution of beige fat to acute-CL-induced metabolic activity is low compared with that of iBAT, even after sustained adrenergic stimulation.

β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia.

BackgroundPerturbed balance between NO and O2 •−. (ie, NO/redox imbalance) is central in the pathobiology of diabetes‐induced vascular dysfunction. We examined whether stimulation of β3 adrenergic receptors (β3 ARs), coupled to endothelial nitric oxide synthase (eNOS) activation, would re‐establish NO/redox balance, relieve oxidative inhibition of the membrane proteins eNOS and Na+‐K+ (NK) pump, and improve vascular function in a new animal model of hyperglycemia.Methods and ResultsWe established hyperglycemia in male White New Zealand rabbits by infusion of S961, a competitive high‐affinity peptide inhibitor of the insulin receptor. Hyperglycemia impaired endothelium‐dependent vasorelaxation by “uncoupling” of eNOS via glutathionylation (eNOS‐GSS) that was dependent on NADPH oxidase activity. Accordingly, NO levels were lower while O2 •− levels were higher in hyperglycemic rabbits. Infusion of the β3 AR agonist CL316243 (CL) decreased eNOS‐GSS, reduced O2 •−, restored NO levels, and improved endothelium‐dependent relaxation. CL decreased hyperglycemia‐induced NADPH oxidase activation as suggested by co‐immunoprecipitation experiments, and it increased eNOS co‐immunoprecipitation with glutaredoxin‐1, which may reflect promotion of eNOS de‐glutathionylation by CL. Moreover, CL reversed hyperglycemia‐induced glutathionylation of the β1 NK pump subunit that causes NK pump inhibition, and improved K+‐induced vasorelaxation that reflects enhancement in NK pump activity. Lastly, eNOS‐GSS was higher in vessels of diabetic patients and was reduced by CL, suggesting potential significance of the experimental findings in human diabetes.Conclusionsβ3 AR activation restored NO/redox balance and improved endothelial function in hyperglycemia. β3 AR agonists may confer protection against diabetes‐induced vascular dysfunction.

Inducible brown adipocytes in subcutaneous inguinal white fat: the role of continuous sympathetic stimulation.

Brown adipocytes (BA) generate heat in response to sympathetic activation and are the main site of nonshivering thermogenesis in mammals. Although most BA are located in classic brown adipose tissue depots, BA are also abundant in the inguinal white adipose tissue (iWAT) before weaning. The number of BA is correlated with the density of sympathetic innervation in iWAT; however, the role of continuous sympathetic tone in the establishment and maintenance of BA in WAT has not been investigated. BA marker expression in iWAT was abundant in weaning mice but was greatly reduced by 8 wk of age. Nonetheless, BA phenotype could be rapidly reinstated by acute β₃-adrenergic stimulation with CL-316,243 (CL). Genetic tagging of adipocytes with adiponectin-CreER(T2) demonstrated that CL reinstates uncoupling protein 1 (UCP1) expression in adipocytes that were present before weaning. Chronic surgical denervation dramatically reduced the ability of CL to induce the expression of UCP1 and other BA markers in the tissue as a whole, and this loss of responsiveness was prevented by concurrent treatment with CL. These results indicate that ongoing sympathetic activity is critical to preserve the ability of iWAT fat cells to express a BA phenotype upon adrenergic stimulation.

1646 EFFECT OF A SELECTIVE β3-ADRENERGIC AGONIST (CL-316,243) ON CYSTOMETRIC PARAMETERS IN CONSCIOUS NORMAL AND SPINAL CORD INJURED FEMALE RATS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Neurogenic Voiding Dysfunction1 Apr 20121646 EFFECT OF A SELECTIVE β3-ADRENERGIC AGONIST (CL-316,243) ON CYSTOMETRIC PARAMETERS IN CONSCIOUS NORMAL AND SPINAL CORD INJURED FEMALE RATS Jean-Baptiste Beauval, Veronique Guilloteau, Monica Capellini, Marc Guerard, Timothy D. Westfall, Xavier Gamé, Stefano Palea, and Philippe Lluel Jean-Baptiste BeauvalJean-Baptiste Beauval Toulouse, France More articles by this author , Veronique GuilloteauVeronique Guilloteau Toulouse, France More articles by this author , Monica CapelliniMonica Capellini Toulouse, France More articles by this author , Marc GuerardMarc Guerard Toulouse, France More articles by this author , Timothy D. WestfallTimothy D. Westfall Toulouse, France More articles by this author , Xavier GaméXavier Gamé Toulouse, France More articles by this author , Stefano PaleaStefano Palea Toulouse, France More articles by this author , and Philippe LluelPhilippe Lluel Toulouse, France More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1478AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In rats with detrusor overactivity secondary to bladder outlet obstruction, β3-adrenergic agonists were shown to increase bladder capacity and decrease non-voiding contractions (NVC). The aim of this study was to determine the effect of CL-316,243 (CL) a selective β3-adrenergic agonist on cystometric parameters in conscious normal rats and to evaluate its effects in a model of bladder overactivity in conscious female rats after spinal cord injury (SCI). METHODS Adult female Wistar rats were used. Cystometry was performed in two different sets of experiments, one in conscious normal animals and the other in conscious rats after SCI at the T8 level for 5 weeks (or sham). Briefly, under anesthesia the jugular vein and urinary bladder were catheterized. 2 days later the bladder was continuously infused with saline. After stabilization (basal cystomeric values), vehicle or CL were administered intravenously. Then, intravesical pressure was recorded for 1 hour. Micturition frequency (MF), intercontraction interval (ICI) and amplitude of micturition (AM) were analyzed and averaged for the 60 min period post-administration. For each parameter, results were compared to baseline. CL was tested at 0.01, 0.03 and 0.1 mg/kg in normal rats and at 0.01 mg/kg in rats with SCI. RESULTS Normal rats. CL significantly increased ICI and decreased MF in a dose dependent manner (-39±4, -46±5, -50±6% at 0.01, 0.03 and 0.1 mg/kg, respectively). No effect on AM was observed with low doses of CL (0.01 and 0.03 mg/kg), while at the highest dose (0.1 mg/kg) AM was significantly decreased (-19±6%). SCI rats. In comparison to sham, SCI rats presented with increased ICI (475±38 vs. 318±53 sec, p=0.025), decreased MF (4±0.3 vs. 8±1.4 pics/30 min, p=0.024), increased AM (p=0.0004) and increased bladder weight (435±39 vs. 147±6 g, p<0.0001). During the filling phase NVCs were also observed. CL (0.01 mg/kg) significantly increased ICI in sham and SCI rats (42±17% and 49±17%, respectively). MF was significantly reduced by 38±10% in SCI rats (p=0.007) while no effect on AM was observed in either group. At this dose, CL specifically reduced the frequency (-53±14%) but not the amplitude of NVCs. CONCLUSIONS This is the first demonstration that stimulation of β3-adrenoceptors produces a decrease in MF and NVCs in SCI rats. These effects are observed at low doses without deleterious effects on AM. Our results suggest that β3-adrenergic agonists could be useful for the treatment of neurogenic bladder overactivity. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e665 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jean-Baptiste Beauval Toulouse, France More articles by this author Veronique Guilloteau Toulouse, France More articles by this author Monica Capellini Toulouse, France More articles by this author Marc Guerard Toulouse, France More articles by this author Timothy D. Westfall Toulouse, France More articles by this author Xavier Gamé Toulouse, France More articles by this author Stefano Palea Toulouse, France More articles by this author Philippe Lluel Toulouse, France More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of electroacupuncture on plasma glucose response to exogenously administered beta3-adrenoceptor agonist in rats

Beta3-adrenoceptor stimulation enhances glucose uptake, lowering plasma glucose concentration. Glucose uptake is also enhanced by muscle contraction. Since electroacupuncture causes stimulated muscles to contract, we studied whether electroacupuncture influences the effect of exogenously administered beta 3 selective adrenoceptor agonist CL-316,243 (CL). Experiments were done in rats anesthetized with urethane and chloralose. Electroacupuncture was delivered for 10min at 10mA, 20Hz to the right tibialis anterior muscle.

Correlation between pharmacologically-induced changes in cystometric parameters and spinal c-Fos expression in rats

The inhibition of bladder sensory transmission is critical for the pharmacotherapy of urine storage symptoms. The purpose of this study is to examine the correlation between pharmacologically-induced changes in cystometric parameters and spinal c-Fos expression in anesthetized rats with bladder hyperactivity induced by the intravesical infusion of acetic acid. Animals were intravenously infused with either oxybutynin (OXY), a muscarinic receptor antagonist, tamsulosin (TAM), an α1-adrenoceptor antagonist, CL316243 (CL), a β3-adrenoceptor agonist, or saline. Morphine (MOR) treatment served as a positive control to inhibit bladder afferent activity. Intermicturition intervals, micturition pressure and pressure threshold were measured after intravesical acetic acid infusion. Animals were then perfused and spinal cords were removed. Sections from the L6 spinal cord were immunostained with an anti-c-Fos antibody, and c-Fos positive cells in the dorsal region were counted. CL and MOR significantly increased intermicturition intervals, whereas OXY and TAM had no significant effect on intermicturition intervals. While TAM and MOR did not affect the micturition pressure, OXY and CL caused a significant decrease. Pressure threshold was significantly decreased by CL and increased by MOR. All drugs significantly decreased the number of c-Fos positive cells with the following order of efficacy: MOR > CL > OXT > TAM. The number of c-Fos positive cells in each animal from all treatment groups was negatively correlated with its average intermicturition interval and pressure threshold, but not with its micturition pressure. Bladder afferent activity is suppressed by several clinically proven mechanisms as measured by c-Fos expression, despite the varied effects on cystometric parameters of each pharmacological treatment.

Role of hormone-sensitive lipase in β-adrenergic remodeling of white adipose tissue

Free fatty acids (FFA) are important extracellular and intracellular signaling molecules and are thought to be involved in beta-adrenergic-induced remodeling of adipose tissue, which involves a transient inflammatory response followed by mitochondrial biogenesis and increased oxidative capacity. This work examined the role of hormone-sensitive lipase (HSL), a key enzyme of acylglycerol metabolism, in white adipose tissue (WAT) remodeling using genetic inactivation or pharmacological inhibition. Acute treatment with the beta(3)-adrenergic agonist CL-316,243 (CL) induced expression of inflammatory markers and caused extravasation of myeloid cells in WAT of wild-type (WT) mice. HSL-knockout (KO) mice had elevated inflammatory gene expression in the absence of stimulation, and acute injection of CL did not further recruit myeloid cells, nor did it further elevate inflammatory gene expression. Acute pharmacological inhibition of HSL with BAY 59-9435 (BAY) had no effect on inflammatory gene expression in WAT or in cultured 3T3-L1 adipocytes. However, BAY prevented induction of inflammatory cytokines by beta-adrenergic stimulation in WAT in vivo and in cultured 3T3-L1 adipocytes. Chronic CL treatment stimulated mitochondrial biogenesis, expanded oxidative capacity, and increased lipid droplet fragmentation in WT mice, and these effects were significantly impaired in HSL-KO mice. In contrast to HSL-KO mice, mice with defective signaling of Toll-like receptor 4, a putative FFA receptor, showed normal beta-adrenergic-induced remodeling of adipose tissue. Overall, results reveal the importance of HSL activity in WAT metabolic plasticity and inflammation.

Indispensable role of mitochondrial UCP1 for antiobesity effect of β3-adrenergic stimulation

Mitochondrial uncoupling protein-1 (UCP1) has been thought to be a key molecule for thermogenesis during cold exposure and spontaneous hyperphagia and thereby in the autonomic regulation of energy expenditure and adiposity. However, UCP1 knockout (KO) mice were reported to be cold intolerant but unexpectedly did not get obese even after hyperphagia, implying that UCP1 may not be involved in the regulation of adiposity. Treatment of obese animals with beta3-adrenergic agonists is known to increase lipid mobilization, induce UCP1, and, finally, reduce body fat content. To obtain direct evidence for the role of UCP1 in the anti-obesity effect of beta3-adrenergic stimulation, in the present study, UCP1-KO and wild-type (WT) mice were fed on cafeteria diets for 8 wk and then given a beta3-adrenergic agonist, CL-316,243 (CL), or saline for 2 wk. A single injection of CL increased whole body oxygen consumption and brown fat temperature in WT mice but not in KO mice, and it elicited almost the same plasma free fatty acid response in WT and KO mice. WT and KO mice increased similarly their body and white fat pad weights on cafeteria diets compared with those on laboratory chow. Daily treatment with CL resulted in a marked reduction of white fat pad weight and the size of adipocytes in WT mice, but not in KO mice. Compared with WT mice, KO mice expressed increased levels of UCP2 in brown fat but decreased levels in white fat and comparable levels of UCP3. It was concluded that the anti-obesity effect of beta3-adrenergic stimulation is largely attributable to UCP1, but less to UCP2 and UCP3, and thereby to UCP1-dependent degradation of fatty acids released from white adipose tissue.