AC5KO Mice Research Articles

Abstract 13767: A Novel Pharmacological Inhibitor of Type 5 Adenylyl Cyclase Inhibitor Enhances Exercise Capacity and Protects Against Glucose Intolerance and Myocardial Ischemia

Disruption of adenylyl cyclase 5 (AC5 KO) is a healthful longevity model; not only do the AC5 KO mice live a third longer than wild type (WT) mice, but are also protected against heart failure, obesity and diabetes and exercise intolerance. To translate to the clinics, we developed a new drug, C90, which inhibits AC5, and examined its effects on cardioprotection in pigs, exercise capacity and glucose tolerance in mice. C90 (30 mg/kg/day) or vehicle were chronically administered to age-matched C57/BL6 male mice via osmotic pump. After baseline exercise, the WT mice, all performing similarly, were divided into two groups; one that received C90 for 14 days and the other receiving vehicle for 14 days. After 14 days of C90 treatment, WT mice exhibited greater exercise capacity reflected by longer running distance (384 ± 27 m vs. 253 ± 16 m, p<0.05) and greater work to exhaustion (18.1 ± 1.5 J vs. 12.4 ± 0.7 J, p<0.05) than mice receiving vehicle. Sensitivity to glucose was measured using a glucose tolerance test in mice. Following 14-days treatment with C90 (30 mg/kg/day), the mice exhibited an overall improvement in glucose tolerance, measured as area under the curve (A.U.) for glucose levels following insulin injection, when compared to vehicle (6414 ± 890 A.U. vs. 9658 ± 1039 A.U., p<0.04). Furthermore, C90-treated mice had a lower fasting glucose level on a regular diet, when compared to vehicle (113 ± 6.5 mg/dl vs. 129 ± 4.2 mg/dl, p<0.05). The cardioprotective effects of C90 were previously demonstrated in mice. In the current study, we investigated the cardioprotective effects of C90 in a porcine model of ischemia/reperfusion (I/R). Ischemia was induced for 60 min coronary artery occlusion, followed by 3-hours reperfusion. C90 (1.2 mg/kg), infused 5 min following the start of the reperfusion led to a decreased infarct size/area at risk (INF/AAR) (21.6 ± 4.3% vs. 36.8 ± 2.9%, p<0.05) when compared to vehicle, with no difference between AAR/left ventricle between the C90 and vehicle groups. In view of its lower level of toxicity than other AC5 inhibitors, C90 is an excellent candidate for clinical development for pharmacological treatment of myocardial ischemia, glucose intolerance and exercise intolerance.

Host genotype and exercise exhibit species-level selection for members of the gut bacterial communities in the mouse digestive system

The mammalian gut microbiome can potentially impact host health and disease state. It is known that the mouse-genome, eating-behavior, and exercise-status promotes higher taxonomic rank-level alterations (e.g. family to phyla-level) of the gut microbiota. Here, host genotype or activity status was investigated to determine if selection of individual bacterial species or strains could be discerned within the murine digestive system. For this study, the fecal bacterial community of adenylyl cyclase 5 knock-out (AC5KO, n = 7) mice or their wild-type (WT, n = 10) littermates under exercise or sedentary conditions were profiled by sequencing rRNA operons. AC5KO mice were chosen since this genotype displays enhanced longevity/exercise capacity and protects against cardiovascular/metabolic disease. Profiling of rRNA operons using the Oxford MinION yielded 65,706 2-D sequences (after size selection of 3.7–5.7 kb) which were screened against an NCBI 16S rRNA gene database. These sequences were binned into 1,566 different best BLAST hits (BBHs) and counted for each mouse sample. Non-metric multidimensional scaling (NMDS) of the gut microbial community demonstrated clustering by physical activity (p = 0.001) but not by host genotype. Additionally, sequence similarity and phylogenetic analysis demonstrated that different bacterial species (closely related to Muribaculum intestinale and Parasutterella excrementihominis) inhabit AC5KO or WT mice depending on activity status. Other bacterial species of the gut microbiota did not follow such patterning (e.g. Turicibacter sanguinis and Turicimonas muris). Our results support the need of improved taxonomic resolution for better characterization of bacterial communities to deepen our understanding of the role of the gut microbiome on host health.

Mechanisms of Gender Differences in Exercise Capacity

The examination of gender differences has been a recent priority of the NIH. It is well documented that female rodents exhibit higher exercise capacity than their male counterparts. We confirmed this in C57 wild‐type(WT) mice, where female mice exhibited greater, p<0.05, maximal exercise capacity for running distance (469 ± 24 m) than age‐matched male WT mice (337 ± 14 m). The first goal was to examine whether these differences persisted with models of enhanced exercise capacity, e.g. exercise training and the adenylyl cyclase type 5 knockout (AC5 KO) mouse. When WT mice underwent chronic exercise‐training, female mice maintained their superiority over males, with females running a 32% greater distance (p<0.05) than age‐matched trained males. However, AC5 KO mice, which demonstrated enhanced exercise capacity, did not exhibit a gender difference, with female AC5 KO mice (695 ± 51 m) running similar distances to male AC5 KO mice (661 ± 45 m). The next goal was to determine mechanisms mediating these differences. After ovariectomy, WT female mice no longer demonstrated enhanced exercise compared with males. Conversely, chronic administration of estrogen to male mice improved their exercise performance and eliminated the gender differences with exercise. Next we investigated nitric oxide (NO), a downstream target of estrogen. Total NO synthase (NOS) activity was higher in female mice compared with male mice in both sedentary (207 ± 14 U/mg vs. 138 ± 4 U/mg, p<0.05) and exercise trained groups (338 ± 42 vs. 193 ± 15 U/mg, p<0.05). Ovariectomized females exhibited nitric oxide levels similar to males (165 ± 11 U/mg). Administration of L‐NAME, resulting in NO blockade, eliminated the gender differences in exercise performance. In AC5 KO mice NOS activity was not different in male and female mice and similar exercise performance in males and females was still observed after treatment with L‐NAME. Thus, both estrogen and nitric oxide are key mechanisms mediating the enhanced exercise performance in female mice.Support or Funding InformationThis study was supported by the National Institute of Health grant R01HL106511This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Disruption of adenylyl cyclase type 5 mimics exercise training.

Exercise training is key to healthful longevity. Since exercise training compliance is difficult, it would be useful to have a therapeutic substitute that mimicked exercise training. We compared the effects of exercise training in wild-type (WT) littermates with adenylyl cyclase type 5 knock out (AC5 KO) mice, a model of enhanced exercise performance. Exercise performance, measured by maximal distance and work to exhaustion, was increased in exercise-trained WT to levels already attained in untrained AC5 KO. Exercise training in AC5 KO further enhanced their exercise performance. The key difference in untrained AC5 KO and exercise-trained WT was the β-adrenergic receptor signaling, which was decreased in untrained AC5 KO compared to untrained WT but was increased in WT with exercise training. Despite this key difference, untrained AC5 KO and exercise-trained WT mice shared similar gene expression, determined by deep sequencing, in their gastrocnemius muscle with 183 genes commonly up or down-regulated, mainly involving muscle contraction, metabolism and mitochondrial function. The SIRT1/PGC-1α pathway partially mediated the enhanced exercise in both AC5 KO and exercise-trained WT mice, as reflected in the reduced exercise responses after administering a SIRT1 inhibitor, but did not abolish the enhanced exercise performance in the AC5 KO compared to untrained WT. Increasing oxidative stress with paraquat attenuated exercise performance more in untrained WT than untrained AC5 KO, reflecting the augmented oxidative stress protection in AC5 KO. Blocking nitric oxide actually reduced the enhanced exercise performance in untrained AC5 KO and trained WT to levels below untrained WT, demonstrating the importance of this mechanism. These results suggest that AC5 KO mice, without exercise training, share similar mechanisms responsible for enhanced exercise capacity with chronic exercise training, most importantly increased nitric oxide, and demonstrate more reserve with the addition of exercise training. A novel feature of the enhanced exercise performance in untrained AC5 KO mice is their decreased sympathetic tone, which is also beneficial to patients with cardiovascular disease.

Adenylyl cyclase-5 in the dorsal striatum function as a molecular switch for the generation of behavioral preferences for cue-directed food choices.

BackgroundBehavioral choices in habits and innate behaviors occur automatically in the absence of conscious selection. These behaviors are not easily modified by learning. Similar types of behaviors also occur in various mental illnesses including drug addiction, obsessive-compulsive disorder, schizophrenia, and autism. However, underlying mechanisms are not clearly understood. In the present study, we investigated the molecular mechanisms regulating unconditioned preferred behaviors in food-choices.ResultsMice lacking adenylyl cyclase-5 (AC5 KO mice), which is preferentially expressed in the dorsal striatum, consumed food pellets nearly one after another in cages. AC5 KO mice showed aversive behaviors to bitter tasting quinine, but they compulsively chose quinine-containing AC5 KO-pellets over fresh pellets. The unusual food-choice behaviors in AC5 KO mice were due to the gain of behavioral preferences for food pellets containing an olfactory cue, which wild-type mice normally ignored. Such food-choice behaviors in AC5 KO mice disappeared when whiskers were trimmed. Conversely, whisker trimming in wildtype mice induced behavioral preferences for AC5 KO food pellets, indicating that preferred food-choices were not learned through prior experience. Both AC5 KO mice and wildtype mice with trimmed whiskers had increased glutamatergic input from the barrel cortex into the dorsal striatum, resulting in an increase in the mGluR1-dependent signaling cascade. The siRNA-mediated inhibition of mGluR1 in the dorsal striatum in AC5 KO mice and wildtype mice with trimmed whiskers abolished preferred choices for AC5 KO food pellets, whereas siRNA-mediated inhibition of mGluR3 glutamate receptors in the dorsal striatum in wildtype mice induced behavioral preferences for AC5 KO food pellets, thus mimicking AC5 KO phenotypes.ConclusionsOur results show that the gain and loss of behavioral preferences for a specific cue-directed option were regulated by specific cellular factors in the dorsal striatum, such that the preferred food choices were switched on when either the mGluR3-AC5 pathway was inactive or the mGluR1 pathway was active, whereas the preferred food-choices were switched off when mGluR1 or its downstream pathway was suppressed. These results identify the AC5 and mGluR system in the dorsal striatum as molecular on/off switches to direct decisions on behavioral preferences for cue-oriented options.

Inhibition of Adenylyl Cyclase Type 5 Prevents l-DOPA-Induced Dyskinesia in an Animal Model of Parkinson's Disease

The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is widely used as a therapeutic choice for the treatment of patients with Parkinson's disease. However, the long-term use of L-DOPA leads to the development of debilitating involuntary movements, called L-DOPA-induced dyskinesia (LID). The cAMP/protein kinase A (PKA) signaling in the striatum is known to play a role in LID. However, from among the nine known adenylyl cyclases (ACs) present in the striatum, the AC that mediates LID remains unknown. To address this issue, we prepared an animal model with unilateral 6-hydroxydopamine lesions in the substantia nigra in wild-type and AC5-knock-out (KO) mice, and examined behavioral responses to short-term or long-term treatment with L-DOPA. Compared with the behavioral responses of wild-type mice, LID was profoundly reduced in AC5-KO mice. The behavioral protection of long-term treatment with L-DOPA in AC5-KO mice was preceded by a decrease in the phosphorylation levels of PKA substrates ERK (extracellular signal-regulated kinase) 1/2, MSK1 (mitogen- and stress-activated protein kinase 1), and histone H3, levels of which were all increased in the lesioned striatum of wild-type mice. Consistently, FosB/ΔFosB expression, which was induced by long-term L-DOPA treatment in the lesioned striatum, was also decreased in AC5-KO mice. Moreover, suppression of AC5 in the dorsal striatum with lentivirus-shRNA-AC5 was sufficient to attenuate LID, suggesting that the AC5-regulated signaling cascade in the striatum mediates LID. These results identify the AC5/cAMP system in the dorsal striatum as a therapeutic target for the treatment of LID in patients with Parkinson's disease.

Similarly Potent Inhibition of Adenylyl Cyclase by P-Site Inhibitors in Hearts from Wild Type and AC5 Knockout Mice

Adenylyl cyclase type 5 (AC5) was described as major cardiac AC isoform. The knockout of AC5 (AC5KO) exerted cardioprotective effects in heart failure. Our study explored the impact of AC5KO on mouse heart AC activities and evaluated putative AC5-selective inhibitors. In cardiac membranes from AC5KO mice, basal AC activity was decreased, while AC stimulation was intact. The putative AC5-selective P-site inhibitors SQ22,536 [9-(tetra-hydro-2-furanyl)-9H-purin-6-amine], vidarabine (9-β-D-arabinosyladenine) and NKY80 [2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone] inhibited recombinant AC5 more potently than AC2 and AC1, but selectivity was only modest (∼4-40-fold). These compounds inhibited cardiac AC from WT and AC5KO mice with similar potencies. In conclusion, AC regulation in AC5KO hearts was unimpaired, questioning the supposed dominant role of AC5 in the heart. Moreover, the AC inhibitors SQ22,536, NKY80 and vidarabine lack adequate selectivity for AC5 and, therefore, do not present suitable tools to study AC5-specific functions.

Abstract LB-337: Protection against tumor growth by disruption of adenylyl cyclase type 5 (AC5).

Abstract Adenylyl cyclase (AC) catalyzes the conversion of ATP to the second messenger cAMP, which in turn activates protein kinase A (PKA). AC/cAMP/PKA signaling pathway is known to mediate tumor growth but the results of previous studies have been controversial. More importantly, little is known about how specific AC isoforms affect tumor growth. Based on our previous data showing that knocking out the isoform AC type 5 in mice (AC5KO) extends longevity and that the major cause of premature mortality in mice is neoplastic diseases, we hypothesized that AC5KO mice can be protected against tumor growth. We previously demonstrated that AC5KO mice are protected against oxidative stress and obesity, which should also provide protection against neoplastic disease. In order to elucidate whether AC5 plays a role in the tumor microenvironment, we initially investigated the growth of B16F10 melanoma in this model. We have found that AC5KO mice exhibit protection against B16F10 melanoma tumor growth (p<0.01). Tumor latency, wet tumor weights and tumor volume were also significantly reduced in AC5KO mice (p<0.01). At day 27, tumor volumes were significantly lower (p<0.01) (AC5KO mice: 500.30±119 vs. WT: 2692.28±508 mm3). Tumors from AC5KO mice had a 68% decrease in cell proliferation index and AC5HET mice had a 37% both with respect to the WT (p<0.01 and p<0.05 respectively). Also, tumors from AC5KO mice displayed a significantly larger apoptotic rate compared to the AC5WT (2.30±0.48% vs. 1.00±0.20 %, p<0.05). Ablation of AC5 in mice also leads to a significantly lower intra-tumor microvessel density (AC5HET: 8±0.4 and AC5KO: 6.2±0.6 compared to WT: 11.5±0.9; p<0.05 and p<0.01).In addition, AC5KO mice had significantly lower VEGF serum levels than WT mice (AC5KO: 2.5±0.1 vs. AC5WT: 3.3±0.2 ng/ml; p<0.05). Tumors in AC5KO mice induced significantly lower angiogenic response than in WT mice, as measured by total vessel length (AC5KO: 58.7±6.2 vs. WT: 100± 8.5, p<0.001). Obesity is a risk factor for the development of breast cancer. In accord with the fact that AC5KO are protected against obesity, we have found that circulating levels of leptin were significantly reduced in AC5KO: 1.99±0.28 vs. WT: 3.23±0.35 ng/ml (p<0.05). In an attempt to elucidate the role of the stromal AC5 isoform and the connection between the AC5KO protection against obesity and tumor incidence; we crossbred AC5 mice with MMTV Her 2 neu mice; a model prone to develop mammary tumors. We have found that AC5KO x MMTV HER-2 neu mice weighed significantly less (p<0.001) and had a significantly lower mammary tumor incidence (p<0.05). All these results together show that understanding the mechanism underlying the role of AC5 in regulating tumor growth and its anti-tumor properties will have a profound impact on its potential therapeutic application as a target for anticancer drug development. Citation Format: Mariana S. De Lorenzo, Wen Y. Chen, Erdene Baljinnyam, Krista La Perle, Sanford P. Bishop, Thomas E. Wagner, Patricio Abarzua, Arnold B. Rabson, Dorothy E. Vatner, Lydia I. Puricelli, Stephen F. Vatner. Protection against tumor growth by disruption of adenylyl cyclase type 5 (AC5). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-337. doi:10.1158/1538-7445.AM2013-LB-337

Abstract 19323: Disruption of Adenylyl Cyclase Type 5, a Novel Target for Obesity, Diabetes and Diabetic Cardiomyopathy

We have previously reported that an adenylyl cyclase type 5 (AC5) deficient mouse model (AC5 knock out, KO) lives one-third longer than wild type (WT) mice. Despite greater daily food intake the AC5KO mice weigh less than WT mice and are protected against cardiac stress. In addition, the AC5KO mice have the same molecular signature of caloric restriction, the most common effective intervention to increase healthy longevity.. Since caloric restriction improves metabolic profiles and protects against insulin resistance, obesity and diabetes, we placed 6-8 week old male AC5KO mice and their corresponding wild type littermates (WTL) controls on a 35.5% high fat diet (HFD) for 100 days. At sacrifice, fat deposits were reduced 36% in AC5KO and cholesterol levels were reduced from 131±9.9 mg/dl to 105±3.1 mg/dl, p<0.05. Indirect calorimetry demonstrated that the AC5KO mice had a 16.7% increase in oxygen consumption during the light cycle (p<0.01) and 29.2% increase during the dark cycle (p<0.01) compared to WTL on HFD, suggesting enhanced metabolic rate as the mechanism for decreased adiposity and body weight. AC5KO mice also displayed improved glucose tolerance with a 32% reduction in area under the curve concomitant with a 111% increase in whole body insulin sensitivity (insulin tolerance test). In parallel, conscious non-stressed euglycemic-hyperinsulinemic clamp studies confirmed the marked enhancement of glucose homeostasis and insulin sensitivity in the AC5KO mice. Moreover, WTL developed reduced cardiac function consistent with diabetic cardiomyopathy following high fat diet feeding (e.g., left ventricular (LV) ejection fraction fell from 71±2% to 53±2%) whereas AC5 KO mice had better preserved LV ejection fraction (61±3%). In addition, AC5 KO mice showed 33% decrease in cardiac fibrosis and 40% decrease in apoptosis compared to WTL on HFD. Thus, reduction of AC5 protects against obesity, diabetes and diabetic cardiomyopathy, suggesting that AC5 is a potential novel therapeutic target for the treatment of obesity, diabetes and associated cardiomyopathy.

Common mechanisms for calorie restriction and adenylyl cyclase type 5 knockout models of longevity

Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared with WT mice. CR applied to AC5 KO results in rapid decrease in body weight, metabolic deterioration, and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle, and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD, and olfactory receptors in both young- and intermediate-age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR mice, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance.

Prevention of heart failure in mice by an antiviral agent that inhibits type 5 cardiac adenylyl cyclase

Despite numerous discoveries from genetically engineered mice, relatively few have been translated to the bedside, mainly because it is difficult to translate from genes to drugs. This investigation examines an antiviral drug, which also has an action to selectively inhibit type 5 adenylyl cyclase (AC5), a pharmaceutical correlate of the AC5 knockout (KO) model, which exhibits longevity and stress resistance. Our objective was to examine the extent to which pretreatment with this drug, adenine 9-β-d-arabinofuranoside (Ara-A), favorably ameliorates the development of heart failure (HF). Ara-A exhibited selective inhibition for AC5 compared with the other major cardiac AC isoform, AC6, i.e., it reduced AC activity significantly in AC5 transgenic (Tg) mice, but not in AC5KO mice and had little effect in either wild-type or AC6Tg mice. Permanent coronary artery occlusion for 3 wk in C57Bl/6 mice increased mortality and induced HF in survivors, as reflected by reduced cardiac function, while increasing cardiac fibrosis. The AC5 inhibitor Ara-A significantly improved all of these end points and also ameliorated chronic isoproterenol-induced cardiomyopathy. As with the AC5KO mice, Ara-A increased mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation. A MEK inhibitor abolished the beneficial effects of the AC5 inhibitor in the HF model, indicating the involvement of the downstream MEK-ERK pathway of AC5. Our data suggest that pharmacological AC5 inhibition may serve as a new therapeutic approach for HF.

Reduction of Adenylyl Cyclase Type 5 Protects Obesity Induced Cardiomyopathy

Since mice with adenylyl cyclase type 5 (AC5) gene disruption eat more than wild type (WT), but weigh less, the goal of this study was to determine if these mice are protected against obesity and the cardiomyopathy induced by a high fat diet (HFD) for 100 days. AC5KO mice gained less weight/kcal consumed than wild type (WT) on HFD, p<0.05, and insulin resistance and glucose tolerance were improved compared to WT on HFD. The HFD induced cardiomyopathy was characterized by reduced, p<0.05, left ventricular (LV) ejection fraction (EF) (53±2.4%) compared with normal diet (ND) (69±2.9%), whereas the reduction in LVEF was less, p<0.05, in AC5KO on HFD (61±3.0%). The cardiomyopathy was also characterized by increased myocardial fibrosis, assessed by picric Sirius red staining, and increased apoptosis, expressed as TUNEL positive cells per mm2. The AC5KO exhibited reduced fibrosis (1.2±0.1%) compared with WT (1.7±0.1%) on HFD, p<0.05, as well as reduced apoptosis (0.5±0.04) compared with WT (0.9±0.1) on HFD, p<0.05. We also examined the effects of pharmacologic inhibition of AC5 with AraAde, an antiviral compound that has AC5 inhibitory effects; weight gain, glucose tolerance, insulin resistance, LV function and fibrosis were all improved compared to vehicle treated mice on HFD. Thus inhibition of AC5 protects against obesity and the cardiomyopathy it induces.

Abstract 18237: Inhibition of Adenylyl Cyclase Type 5 Protects Against Obesity and Diabetes

Adenylyl cyclase type 5 (AC5) is one of 9 major isoforms of AC. Mice with AC5 gene disruption (knockout, KO) live one third longer than wild type (WT) littermates. Like caloric restriction, the most commonly studied model of longevity, the AC5KO mice weigh 12% less than WT (p<0.05) and have less body fat, i.e., adiposity index, calculated by dividing the sum weights of the three major fat pads (inguinal, retroperitoneal, gonadal) by the body weight, was 50% lower than WT (p<0.05). The first goal of this study was to determine if AC5KO mice are protected, not only against obesity, but also diabetes. Insulin sensitivity, assessed by blood glucose measurement following insulin injection, was improved by 24% compared to WT. Glucose tolerance was also improved, p<0.05. A major limitation of studies in genetically altered mouse models, is that it is difficult to translate these results to the bedside, since it is not possible to knock out a gene in patients. Accordingly, we identified a pharmacological inhibitor of AC5, vidaribine, and examined the extent to which it ameliorated obesity and diabetes. First we demonstrated its selectivity for AC5; the inhibitor reduced inotropic responses to isoproterenol more than WT only in AC5 cardiac transgenic mice, but not in AC6 cardiac transgenic mice. To induce obesity, WT mice were fed a high fat diet feeding for 2 months, increasing body weight by 51% over mice fed a standard diet. WT mice with high fat diet and AC5 inhibitor on board for an additional 3 months, compared with WT mice on vehicle and high fat diet, increased body weight and adiposity index less, p<0.05, by 20% and 28%, respectively, while insulin resistance was reduced by 76% and glucose tolerance was improved by 66% (p<0.05). In AC5KO mice on the high fat diet insulin resistance was also reduced and glucose tolerance was also improved compared to WT on the high fat diet, similar to that observed with the AC5 inhibitor on the high fat diet. Thus, inhibition of AC5 provides a novel target for obesity and diabetes; the effective pharmacological inhibitor of AC5 enhances its clinical relevance.

Down‐regulation of MnSOD via Sirt1/FoxO3a complex increase oxidative stress with cardiac overexpression of Type 5 Adenylyl Cyclase

We examined the stress of chronic (7 days) isoproterenol (ISO) stimulation 60mg/kg/day in 3–5 month AC5 Tg mice and WT littermates. Over 30% Tg mice died after day 3 while none of the WT mice died (n=12/group). Oxidative stress induced by chronic ISO stimulation was detected by 8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) staining and AC5 Tg hearts showed 27% more positive staining than WT. To further examine whether AC5 Tg mice are less tolerant to oxidative stress, we injected paraquat (PQ), an oxidative stress inducer, and again more positive 8‐OHdG staining was observed in AC5 Tg mice hearts than AC5 KO mice, while the anti‐oxidant MnSOD protein was 40% lower in AC5 Tg than WT. We further hypothesized that inhibition of AC5 activates Sirt1 and FoxO3a, key transcription regulators of MnSOD. Luciferase test showed 3 fold higher FoxO3a transcriptional activity in AC5 knockdown myocytes. Protein expression of MnSOD was 40% decreased in neonatal myocytes infected by both ad‐AC5‐shRNA and ad‐Sirt1‐shRNA compared with AC5 knockdown myocytes. These data suggest that AC5 inhibits the Sirt1/FoxO3a complex thus down‐regulating MnSOD expression, and consequently increasing oxidative stress.

A molecular dissociation between cued and contextual appetitive learning

In appetitive Pavlovian learning, animals learn to associate discrete cues or environmental contexts with rewarding outcomes, and these cues and/or contexts can potentiate an ongoing instrumental response for reward. Although anatomical substrates underlying cued and contextual learning have been proposed, it remains unknown whether specific molecular signaling pathways within the striatum underlie one form of learning or the other. Here, we show that while the striatum-enriched isoform of adenylyl cyclase (AC5) is required for cued appetitive Pavlovian learning, it is not required for contextual appetitive learning. Mice lacking AC5 (AC5KO) could not learn an appetitive Pavlovian learning task in which a discrete signal light predicted reward delivery, yet they could form associations between context and either natural or drug reward, which could in turn elicit Pavlovian approach behavior. However, unlike wild-type (WT) mice, AC5KO mice could not use these Pavlovian conditioned stimuli to potentiate ongoing instrumental behavior in a Pavlovian-to-instrumental transfer paradigm. These data suggest that AC5 is specifically required for learning associations between discrete cues and outcomes in which the temporal relationship between conditioned stimulus (CS) and unconditioned stimulus (US) is essential, while alternative signaling mechanisms may underlie the formation of associations between context and reward. In addition, loss of AC5 compromises the ability of both contextual and discrete cues to modulate instrumental behavior.

A cAMP Pathway Underlying Reward Prediction in Associative Learning

In associative learning, animals learn to associate external cues or their own actions with appetitive or aversive outcomes. Although the dopamine (DA) system and the striatum/nucleus accumbens have been implicated in both the pavlovian and instrumental form of associative learning, whether specific neuronal signaling mechanisms underlie one form or the other is unknown. Here, we report that the striatum-enriched isoform of adenylyl cyclase (AC), AC5, is selectively required for appetitive pavlovian learning. Mice with genetic deletion of AC5 (AC5KO) acquired instrumental responding yet were unable to use cues that predicted reward delivery. The specificity of this deficit was confirmed by an inability of AC5KO mice to learn a simple appetitive pavlovian conditioning task. Conversely, AC5KO mice showed intact aversive pavlovian learning, suggesting the deficit was specific for learning about appetitive outcomes. Our results suggest that AC5 is a critical component of DA-dependent strengthening of stimulus-reward contingencies.

Adenylyl Cyclase Type 5 Disruption Prolongs Longevity and Protects the Heart against Stress

We tested the hypothesis that longevity and stress resistance are mediated by common mechanisms. We have recently identified a novel, genetically engineered animal model, i.e., mice with adenylyl cyclase (AC) type 5 “knocked out” (AC5 KO), which live 30% longer than wild type (WT) animals and do not exhibit the cardiomyopathy and osteoporosis of old age. Thoracic aortic banding reduced left ventricular (LV) ejection fraction (LVEF) in WT (from 70 ± 2.8 to 57 ± 3.9%, P < 0.05), but this was not observed in AC5 KO mice. Chronic (1–2 wks) isoproterenol (ISO) infusion induced more effective desensitization in AC5 KO and less apoptosis. Mice with cardiac overexpression of either β1-adrenergic receptors (β1-AR Tg) or β2-adrenergic receptors (β2-AR Tg) exhibit the opposite pattern, i.e. development of cardiomyopathy and enhanced premature mortality and less resistance to stress with either chronic pressure overload or chronic catecholamine stimulation. Mating the AC5 KO with β-AR Tg mice rescued their cardiomyopathy. Thus, AC5 is an important mechanism regulating lifespan and stress resistance and protects the heart against stress and from the cardiomyopathy induced by chronically enhanced β-AR signaling. Since the mechanisms of stress resistance appear to involve more effective desensitization, inhibition of AC5 could be a novel approach to the therapy of heart failure.

Calcium-Sensitive Adenylyl Cyclases in Depression and Anxiety: Behavioral and Biochemical Consequences of Isoform Targeting

Adenylyl cyclases (ACs) represent a diverse family of enzymes responsible for the generation of cyclic adenosine monophosphate (cAMP), a key intracellular second messenger. The Ca(2+)/calmodulin-stimulated AC1 and AC8 isoforms as well as the calcium-inhibited AC5 isoform are abundantly expressed within limbic regions of the central nervous system. This study examines the contribution of these AC isoforms to emotional behavior. Male and female AC1/8 double knockout mice (DKO) and AC5 knockout mice (AC5KO) were examined on a series of standard laboratory assays of emotionality. Mice were also assayed for hippocampal cell proliferation and for changes in brain-derived neurotrophic factor signaling in the nucleus accumbens, amygdala, and hippocampus, three forebrain structures involved in the regulation of mood and affect. The AC5KO mice showed striking anxiolytic and antidepressant phenotypes on standard behavioral assays. In contrast, AC1/8 DKO mice were hypoactive, exhibited diminished sucrose preference, and displayed alterations in neurotrophic signaling, generally consistent with a prodepressant phenotype. Neither line of mice displayed alterations in hippocampal cell proliferation. These data illustrate the complex manner in which Ca(2+)/calmodulin-stimulated ACs contribute to emotional behavior. In addition, they support the possibility that a selective AC5 antagonist would be of therapeutic value against depression and anxiety disorders.

Type 5 Adenylyl Cyclase Disruption Increases Longevity and Protects Against Stress

Mammalian models of longevity are related primarily to caloric restriction and alterations in metabolism. We examined mice in which type 5 adenylyl cyclase (AC5) is knocked out (AC5 KO) and which are resistant to cardiac stress and have increased median lifespan of approximately 30%. AC5 KO mice are protected from reduced bone density and susceptibility to fractures of aging. Old AC5 KO mice are also protected from aging-induced cardiomyopathy, e.g., hypertrophy, apoptosis, fibrosis, and reduced cardiac function. Using a proteomic-based approach, we demonstrate a significant activation of the Raf/MEK/ERK signaling pathway and upregulation of cell protective molecules, including superoxide dismutase. Fibroblasts isolated from AC5 KO mice exhibited ERK-dependent resistance to oxidative stress. These results suggest that AC is a fundamentally important mechanism regulating lifespan and stress resistance.