Modification of cardiac disease by transgenically altered histone deacetylase 6.

Histone deacetylase 6 (HDAC6) is known to deacetylate amino acid lysine in alpha-tubulin. However, the functional role of HDAC6 in the progression of cardiac disease remains uncertain. The functional role of HDAC6 in the hearts was examined using transgenic (TG) mice expressing either human wild-type HDAC6, deacetylase inactive HDAC6 (HDAC6H216A, H611A), and human HDAC6 replaced all serine or threonine residues with aspartic acid at N-terminal 1- 43 amino acids (HDAC6NT-allD) specifically in the hearts. Overexpression of wild-type HDAC6 significantly reduced acetylated tubulin levels, and overexpression of HDAC6H216A, H611A significantly increased it in the mouse hearts. Detectable acetylated tubulin disappeared in HDAC6NT-allD TG mouse hearts. Neither histological alteration nor alteration of cardiac function was observed in the HDAC6 TG mouse hearts. To analyze the role of HDAC6 and acetylated tubulin in disease conditions, we examined HDAC6 in isoprenaline-induced hypertrophy or pressure-overload hypertrophy (TAC). No obvious alteration in the heart weight/body weight ratio or gene expressions of hypertrophic markers between NTG and HDAC6NT-allD mice was observed following treatment with isoprenaline. In contrast, a marked reduction in the shortening fraction and dilated chamber dilatation was detected in the HDAC6NT-allD TG mouse hearts 2 weeks after TAC. A sustained low level of acetylated tubulin and acetylated cortactin was observed in the TAC HDAC6NT-allD TG mouse hearts. Cardiac HDAC6 activity that can regulate acetylated levels of tubulin and cortactin may be critical factors involved in cardiac disease such as pressure-overload hypertrophy.

mTOR-mediated calcium transients affect cardiac function in ex vivo ischemia-reperfusion injury.

The mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, cell growth, and survival. While previous studies using transgenic mice with cardiac-specific overexpression of mTOR (mTOR-Tg) demonstrated the protective effects of cardiac mTOR against ischemia-reperfusion (I/R) injury in both ex vivo and in vivo models, the mechanisms underlying the role of cardiac mTOR in cardiac function following I/R injury are not well-understood. Torin1, a pharmacological inhibitor of mTOR complex (mTORC) 1 and mTORC2, significantly decreased functional recovery of LV developed pressure in ex vivo I/R models (p < 0.05). To confirm the role of mTOR complexes in I/R injury, we generated cardiac-specific mTOR-knockout (CKO) mice. In contrast to the effects of Torin1, CKO hearts recovered better after I/R injury than control hearts (p < 0.05). Interestingly, the CKO hearts had exhibited irregular contractions during the reperfusion phase. Calcium is a major factor in Excitation-Contraction (EC) coupling via Sarcoplasmic Reticulum (SR) calcium release. Calcium is also key in opening the mitochondrial permeability transition pore (mPTP) and cell death following I/R injury. Caffeine-induced SR calcium release in isolated CMs showed that total SR calcium content was lower in CKO than in control CMs. Western blotting showed that a significant amount of mTOR localizes to the SR/mitochondria and that GSK3-ß phosphorylation, a key factor in SR calcium mobilization, was decreased. These findings suggest that cardiac mTOR located to the SR/mitochondria plays a vital role in EC coupling and cell survival in I/R injury.

Cardiac mTOR rescues the detrimental effects of diet-induced obesity in the heart after ischemia-reperfusion.

Diet-induced obesity deteriorates the recovery of cardiac function after ischemia-reperfusion (I/R) injury. While mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, the effects of cardiac mTOR in ischemic injury under metabolic syndrome remains undefined. Using cardiac-specific transgenic mice overexpressing mTOR (mTOR-Tg mice), we studied the effect of mTOR on cardiac function in both ex vivo and in vivo models of I/R injury in high-fat diet (HFD)-induced obese mice. mTOR-Tg and wild-type (WT) mice were fed a HFD (60% fat by calories) for 12 wk. Glucose intolerance and insulin resistance induced by the HFD were comparable between WT HFD-fed and mTOR-Tg HFD-fed mice. Functional recovery after I/R in the ex vivo Langendorff perfusion model was significantly lower in HFD-fed mice than normal chow diet-fed mice. mTOR-Tg mice demonstrated better cardiac function recovery and had less of the necrotic markers creatine kinase and lactate dehydrogenase in both feeding conditions. Additionally, mTOR overexpression suppressed expression of proinflammatory cytokines, including IL-6 and TNF-α, in both feeding conditions after I/R injury. In vivo I/R models showed that at 1 wk after I/R, HFD-fed mice exhibited worse cardiac function and larger myocardial scarring along myofibers compared with normal chow diet-fed mice. In both feeding conditions, mTOR overexpression preserved cardiac function and prevented myocardial scarring. These findings suggest that cardiac mTOR overexpression is sufficient to prevent the detrimental effects of diet-induced obesity on the heart after I/R, by reducing cardiac dysfunction and myocardial scarring.

Three-dimensional myocardial scarring along myofibers after coronary ischemia-reperfusion revealed by computerized images of histological assays.

Adverse left ventricular (LV) remodeling after acute myocardial infarction is characterized by LV dilatation and development of a fibrotic scar, and is a critical factor for the prognosis of subsequent development of heart failure. Although myofiber organization is recognized as being important for preserving physiological cardiac function and structure, the anatomical features of injured myofibers during LV remodeling have not been fully defined. In a mouse model of ischemia-reperfusion (I/R) injury induced by left anterior descending coronary artery ligation, our previous histological assays demonstrated that broad fibrotic scarring extended from the initial infarct zone to the remote zone, and was clearly demarcated along midcircumferential myofibers. Additionally, no fibrosis was observed in longitudinal myofibers in the subendocardium and subepicardium. However, a histological analysis of tissue sections does not adequately indicate myofiber injury distribution throughout the entire heart. To address this, we investigated patterns of scar formation along myofibers using three-dimensional (3D) images obtained from multiple tissue sections from mouse hearts subjected to I/R injury. The fibrotic scar area observed in the 3D images was consistent with the distribution of the midcircumferential myofibers. At the apex, the scar formation tracked along the myofibers in an incomplete C-shaped ring that converged to a triangular shape toward the end. Our findings suggest that myocyte injury after transient coronary ligation extends along myofibers, rather than following the path of coronary arteries penetrating the myocardium. The injury pattern observed along myofibers after I/R injury could be used to predict prognoses for patients with myocardial infarction.

Systemic PPARγ deletion impairs circadian rhythms of behavior and metabolism.

Compelling evidence from both human and animal studies suggests a physiological link between the circadian rhythm and metabolism but the underlying mechanism is still incompletely understood. We examined the role of PPARγ, a key regulator of energy metabolism, in the control of physiological and behavioral rhythms by analyzing two strains of whole-body PPARγ null mouse models. Systemic inactivation of PPARγ was generated constitutively by using Mox2-Cre mice (MoxCre/flox) or inducibly by using the tamoxifen system (EsrCre/flox/TM). Circadian variations in oxygen consumption, CO(2) production, food and water intake, locomotor activity, and cardiovascular parameters were all remarkably suppressed in MoxCre/flox mice. A similar phenotype was observed in EsrCre/flox/TM mice, accompanied by impaired rhythmicity of the canonical clock genes in adipose tissues and liver but not skeletal muscles or the kidney. PPARγ inactivation in isolated preadipocytes following exposure to tamoxifen led to a similar blockade of the rhythmicity of the clock gene expression. Together, these results support an essential role of PPARγ in the coordinated control of circadian clocks and metabolic pathways.

Cardiac mTOR protects the heart against ischemia-reperfusion injury.

Cardiac mammalian target of rapamycin (mTOR) is necessary and sufficient to prevent cardiac dysfunction in pathological hypertrophy. However, the role of cardiac mTOR in heart failure after ischemic injury remains undefined. To address this question, we used transgenic (Tg) mice with cardiac-specific overexpression of mTOR (mTOR-Tg mice) to study ischemia-reperfusion (I/R) injury in two animal models: 1) in vivo I/R injury with transient coronary artery ligation and 2) ex vivo I/R injury in Langendorff-perfused hearts with transient global ischemia. At 28 days after I/R, mortality was lower in mTOR-Tg mice than littermate control mice [wild-type (WT) mice]. Echocardiography and MRI demonstrated that global cardiac function in mTOR-Tg mice was preserved, whereas WT mice exhibited significant cardiac dysfunction. Masson's trichrome staining showed that 28 days after I/R, the area of interstitial fibrosis was smaller in mTOR-Tg mice compared with WT mice, suggesting that adverse left ventricular remodeling is inhibited in mTOR-Tg mice. In the ex vivo I/R model, mTOR-Tg hearts demonstrated improved functional recovery compared with WT hearts. Perfusion with Evans blue after ex vivo I/R yielded less staining in mTOR-Tg hearts than WT hearts, indicating that mTOR overexpression inhibited necrosis during I/R injury. Expression of proinflammatory cytokines, including IL-6 and TNF-α, in mTOR-Tg hearts was lower than in WT hearts. Consistent with this, IL-6 in the effluent post-I/R injury was lower in mTOR-Tg hearts than in WT hearts. These findings suggest that cardiac mTOR overexpression in the heart is sufficient to provide substantial cardioprotection against I/R injury and suppress the inflammatory response.

Phosphoinositide-3 kinase signaling in cardiac hypertrophy and heart failure.

Heart failure, a major symptom in the progression of cardiac hypertrophy, is a critical risk factor for cardiac death. A large body of research has investigated cardioprotective mechanisms that prevent or minimize hypertrophy, identifying a variety of specific peptide hormones, growth factors, and cytokines with cardioprotective properties. Recent investigation of the downstream effector pathways for these growth factors has identified molecules involved in the progression of cardiac hypertrophy and heart failure, including phosphoinositide 3-kinase (PI3K), Akt and mammalian target of rapamycin (mTOR). Using genetically modified transgenic or knockout mice and adenoviral targeting to manipulate expression or function in experimental models of heart failure, several investigators have demonstrated that the PI3K-Akt pathway regulates cardiomyocyte size, survival, angiogenesis, and inflammation in both physiological and pathological cardiac hypertrophy. In this review, we discuss the reciprocal regulation of PI3K, Akt and mTOR in cardiomyocytes and their association with cardiac disease.

Enhanced glucose tolerance in the Brattleboro rat.

[Arg(8)]-vasopressin (AVP) plays a crucial role in regulating body fluid retention, which is mediated through the vasopressin V(2) receptor in the kidney. In addition, AVP is involved in the regulation of glucose homeostasis via vasopressin V(1A) and vasopressin V(1B) receptors. Our previous studies demonstrated that vasopressin V(1A) receptor-deficient (V(1A)R-/-) and V(1B) receptor-deficient (V(1B)R-/-) mice exhibited hyperglycemia and hypoglycemia with hypoinsulinemia, respectively. These findings indicate that vasopressin V(1A) receptor deficiency results in decreased insulin sensitivity whereas vasopressin V(1B) receptor deficiency results in increased insulin sensitivity. In addition, vasopressin V(1A) and vasopressin V(1B) receptor double-deficient (V(1AB)R-/-) mice exhibited impaired glucose tolerance, suggesting that the effects of vasopressin V(1B) receptor deficiency do not influence the development of hyperglycemia promoted by vasopressin V(1A) receptor deficiency, and that the blockage of both receptors could lead to impaired glucose tolerance. However, the contributions of the entire AVP/vasopressin receptors system to the regulation of blood glucose have not yet been clarified. In this study, to further understand the role of AVP/vasopressin receptors signaling in blood glucose regulation, we assessed the glucose tolerance of AVP-deficient homozygous Brattleboro (di/di) rats using an oral glucose tolerance test (GTT). Plasma glucose and insulin levels were consistently lower in homozygous di/di rats than in heterozygous di/+ rats during the GTT, suggesting that the blockage of all AVP/vasopressin receptors resulting from the AVP deficiency could lead to enhanced glucose tolerance.

Amelioration of cisplatin nephrotoxicity by genetic or pharmacologic blockade of prostaglandin synthesis.

Nephrotoxicity is a common complication of cisplatin chemotherapy that limits its clinical use. Here, we determined whether arachidonic acid metabolism has a role in the pathogenesis of cisplatin nephrotoxicity in mice. Three days following cisplatin injection, wild-type mice displayed renal functional and structural abnormalities consistent with nephrotoxicity accompanied by elevated circulating and renal levels of TNF-α and renal levels of IL-1ß, subunits of NADPH oxidase, thiobarbituric acid-reactive substances, and PGE(2). These indices of kidney injury, inflammation, oxidative stress, and arachidonate metabolism were all diminished in microsomal prostaglandin E synthase-1 (mPGES-1) null mice; a phenotype recapitulated by treatment of wild-type mice with the COX-2 inhibitor celecoxib. Following cisplatin administration, there was paralleled induction of COX-2 and mPGES-1 in renal parenchymal cells. Interestingly, mPGES-1 null mice were not protected from acute kidney injury caused by ischemia-reperfusion or endotoxin. Hence, our results suggest the activation of COX-2/mPGES-1 pathway in renal parenchymal cells may selectively mediate cisplatin-induced renal injury. This may offer a novel therapeutic target for management of the adverse effect of cisplatin chemotherapy.

The Cardiomyocyte as a Source of Cytokines in Cardiac Injury.

Fibrosis induced by prolonged inflammation is a major pathophysiological feature of adverse left ventricular remodeling after myocardial infarction and pathological cardiac hypertrophy. Recent reports strongly suggest that the interaction between leukocytes, non-myocytes (mainly cardiac fibroblasts) and cardiomyocytes, possibly mediated by cytokine signaling, plays an important role in controlling the inflammatory reaction after cardiac injury. Therefore, controlling cytokine secretion from resident cardiomyocytes is one plausible strategy for preventing tissue damage.

mPGES-1 deletion impairs aldosterone escape and enhances sodium appetite.

Aldosterone (Aldo) is a major sodium-retaining hormone that reduces renal sodium excretion and also stimulates sodium appetite. In the face of excess Aldo, the sodium-retaining action of this steroid is overridden by an adaptive regulatory mechanism, a phenomenon termed Aldo escape. The underlying mechanism of this phenomenon is not well defined but appeared to involve a number of natriuretic factors such prostaglandins (PGs). Here, we investigated the role of microsomal prostaglandin E synthase-1 (mPGES-1) in the response to excess Aldo. A 14-day Aldo infusion at 0.35 mg x kg(-1) x day(-1) via an osmotic minipump in conjunction with normal salt intake did not produce obvious disturbances in fluid metabolism in WT mice as suggested by normal sodium and water balance, plasma sodium concentration, hematocrit, and body weight, despite the evidence of a transient sodium accumulation on days 1 or 2. In a sharp contrast, the 14-day Aldo treatment in mPGES-1 knockoute (KO) mice led to increased sodium and water balance, persistent reduction of hematocrit, hypernatremia, and body weight gain, all evidence of fluid retention. The escaped wild-type (WT) mice displayed a remarkable increase in urinary PGE(2) excretion in parallel with coinduction of mPGES-1 in the proximal tubules, accompanied by a remarkable, widespread downregulation of renal sodium and water transporters. The increase in urinary PGE(2) excretion together with the downregulation of renal sodium and water transporters were all significantly blocked in the KO mice. Interestingly, compared with WT controls, the KO mice exhibited consistent increases in sodium and water intake during Aldo infusion. Together, these results suggest an important role of mPGES-1 in antagonizing the sodium-retaining action of Aldo at the levels of both the central nervous system and the kidney.

mPGES-1 protects against DOCA-salt hypertension via inhibition of oxidative stress or stimulation of NO/cGMP.

Microsomal prostaglandin E synthase-1 (mPGES-1) is a recently characterized cytokine-inducible enzyme critically involved in pain and inflammatory response. However, its role in blood pressure regulation is still debatable. The present study was undertaken to examine the effect of mPGES-1 deletion on DOCA-salt hypertension. After 2 weeks of DOCA plus 1% NaCl as drinking fluid, hypertension and sodium retention were more severe in mPGES-1 knockout (KO) mice than in wild-type (WT) controls. The indices of oxidative stress including urinary 8-isprostane and renal thiobarbituric acid-reactive substances were only modestly increased or unchanged in the WT mice but more significantly increased in the KO mice after DOCA-salt. Conversely, in response to DOCA-salt, the indices of antioxidant systems including renal expression of superoxide dismutase-3 and urinary nitrate/nitrite excretion were all significantly elevated in the WT mice but remarkably suppressed in the KO mice. Tempol treatment (50 mg/kg per day) in DOCA-salt KO mice produced a marked attenuation of hypertension, sodium retention, and kidney injury. Immunoblotting demonstrated increased renal expression of mPGES-1 in DOCA-salt WT mice. DOCA-salt induced a nearly 5-fold increase in urinary PGE(2) excretion in the WT mice, and this increase was completely abolished in the KO mice. Together, these results suggest that mPGES-1-derived PGE(2) confers protection against DOCA-salt hypertension likely via inhibition of oxidative stress or stimulation of superoxide dismutase-3 and urinary nitrate/nitrite system.

Enhanced effect of neuropeptide Y on food intake caused by blockade of the V(1A) vasopressin receptor.

Food intake is regulated by various factors such as neuropeptide Y. Neuropeptide Y potently induces an increase in food intake, and simultaneously stimulates arginine-vasopressin (AVP) secretion in the brain. Recently, we reported that V(1A) vasopressin receptor-deficient (V(1A)R(-/-)) mice exhibited altered daily food intake accompanied with hyperglycemia and hyperleptinemia. Here, we further study the involvement of the AVP/V(1A) receptor in the appetite regulation of neuropeptide Y with V(1A)R(-/-) mice and antagonists for the AVP receptor. The intra-cerebral-ventricle administration of neuropeptide Y induced greater food consumption in V(1A)R(-/-) mice than wild-type (WT) mice, whereas an anorexigenic effect of leptin was not different between the two groups. This finding suggests that the orexigenic effect of neuropeptide Y was enhanced in V(1A)R(-/-) mice, leading to the increased food intake in response to the neuropeptide Y stimulation. In addition, the neuropeptide Y-induced orexigenic effect was enhanced by co-administration of OPC-21268, an antagonist for the V(1A) vasopressin receptor, into the cerebral ventricle in WT mice, whereas the neuropeptide Y-induced orexigenic effect was not affected by co-administration of SSR-149415, an antagonist for the V(1B) vasopressin receptor. These results indicate that AVP could suppress the neuropeptide Y-induced orexigenic effect via the V(1A) vasopressin receptor, and that blockade or inhibition of the AVP/V(1A) receptor signal resulted in the enhanced neuropeptide Y-induced orexigenic effect. Thus, we show that the AVP/V(1A) receptor is involved in appetite regulation as an anorexigenic factor for the neuropeptide Y-induced orexigenic effect.

Vasopressin regulation of blood pressure and volume: findings from V1a receptor-deficient mice.

[Arg(8)]-vasopressin (AVP) has several functions via its three distinct receptors, V1a, V1b, and V2. The V1a vasopressin receptor (V1aR) is expressed in blood vessels and involved in vascular contraction. Recently, we generated V1a receptor-deficient (V1aR(-/-)) mice and found that they were hypotensive. In addition, V1aR(-/-) mice exhibited (1) blunted AVP-induced vasopressor response, (2) impaired arterial baroreceptor reflex, (3) decreased sympathetic nerve activity, and (4) decreased blood volume, all of which could contribute to the observed hypotension. In relation to their decreased blood volume, V1aR(-/-) mice had decreased plasma aldosterone levels, which could result not only from decreased activity of the renin-angiotensin system (RAS), but also from impaired AVP-stimulated aldosterone release in the adrenal glands. V1aR was found to specifically co-express at the macula densa cells with cyclooxygenase (COX)-2 and with neuronal nitric oxide synthase, which produces potent stimulators of renin, PGE(2), and NO. The expression levels of renin, COX-2, and nNOS were significantly decreased in V1aR(-/-) mice, which led to the suppression of RAS activity and consequent decreases in aldosterone and blood volume. Furthermore, V1aR is also expressed in collecting duct cells and involved in regulating water reabsorption by affecting V2/aquaporin 2 function. Thus, AVP regulates blood pressure and volume via V1aR by exerting diverse functions in vivo.

Both V(1A) and V(1B) vasopressin receptors deficiency result in impaired glucose tolerance.

[Arg(8)]-vasopressin (AVP) is involved in the regulation of glucose homeostasis via vasopressin V(1A) and vasopressin V(1B) receptor. Our previous studies have demonstrated that vasopressin V(1A) receptor deficient (V(1A)R(-/-)) mice exhibited hyperglycemia, vasopressin V(1B) receptor deficient (V(1B)R(-/-)) mice, in contrast, exhibited hypoglycemia with hypoinsulinemia. These findings indicate that vasopressin V(1A) receptor deficiency results in decreased insulin sensitivity, whereas vasopressin V(1B) receptor deficiency results in increased insulin sensitivity. In our previous and present studies, we used the glucose tolerance test to investigate glucose tolerance in mutant mice, lacking either the vasopressin V(1A) receptor, the vasopressin V(1B) receptor, or both receptors, that were kept on a high-fat diet. Glucose and insulin levels were lower in V(1B)R(-/-) mice than in wild type (WT) mice when both groups were fed the high-fat diet, which indicates that the insulin sensitivity of the V(1B)R(-/-) mice was enhanced. V(1A)R(-/-) mice on the high-fat diet, on the other hand, exhibited overt obesity, along with an impaired glucose tolerance, while WT mice on the high-fat diet did not. Next, in order to assess the effect of vasopressin V(1B) receptor deficiency on the development of glucose intolerance caused by vasopressin V(1A) receptor deficiency, we generated mice that were deficient for both vasopressin V(1A) receptor and vasopressin V(1B) receptor (V(1AB)R(-/-)), fed them a high-fat diet, and examined their glucose tolerances using the glucose tolerance test. Glucose tolerance was impaired in V(1AB)R(-/-) mice, suggesting that the effects of vasopressin V(1B) receptor deficiency could not influence the development of hyperglycemia promoted by vasopressin V(1A) receptor deficiency, and that blockade of both receptors could lead to impaired glucose tolerance.

Altered lipid metabolism in vasopressin V1B receptor-deficient mice.

We previously reported that insulin sensitivity was increased in vasopressin V(1B) receptor-deficient (V(1B)R(-/-)) mice. Here, we investigate the lipid metabolism in V(1B)R(-/-) mice. Despite having lower body weight, V(1B)R(-/-) mice had significantly greater fat weight of the epididymal white adipose tissue than V(1B)R(+/+) mice. Glycerol production and beta-oxidation were suppressed in V(1B)R(-/-) mice under a fasting condition, and isoproterenol-stimulated lipolysis in differentiated adipocytes was significantly decreased in V(1B)R(-/-) mice. These results indicated that lipolysis was inhibited in V(1B)R(-/-) mice. On the other hand, lipogenesis was promoted by the increased metabolism from glucose to lipid. Furthermore, our in vivo and in vitro analyses showed that the secretion of adiponectin was increased in V(1B)R(-/-) mice, while the serum leptin level was lower in V(1B)R(-/-) mice. These findings indicated that the insulin sensitivity and lipid metabolism were altered in V(1B)R(-/-) mice and that the increased insulin sensitivity could contribute to the suppressed lipolysis and enhanced lipogenesis, which consequently resulted in the increased fat weight in V(1B)R(-/-) mice.

Vascular PPARgamma controls circadian variation in blood pressure and heart rate through Bmal1.

Thiazolidinediones (TZDs) are PPARgamma activators that exhibit vasculoprotective properties. To determine the vascular function of PPARgamma, we analyzed Tie2Cre/flox and SM22Cre/flox mice. Unexpectedly, both knockout strains exhibited a significant reduction of circadian variations in blood pressure and heart rate in parallel with diminished variations in urinary norepinephrine/epinephrine excretion and impaired rhythmicity of the canonical clock genes, including Bmal1. PPARgamma expression in the aorta exhibited a robust rhythmicity with a more than 20-fold change during the light/dark cycle. Rosiglitazone treatment induced aortic expression of Bmal1 mRNA, and ChIP and promoter assays revealed that Bmal1 is a direct PPARgamma target gene. These studies have uncovered a role for vascular PPARgamma as a peripheral factor participating in regulation of cardiovascular rhythms.

Vasopressin regulates the renin-angiotensin-aldosterone system via V1a receptors in macula densa cells.

The neuropeptide hormone arginine-vasopressin (AVP) is well known to exert its antidiuretic effect via the vasopressin V2 receptor (V2R), whereas the role of the vasopressin V1a receptor (V1aR) in the kidney remains to be clarified. Previously, we reported decreased plasma volume and blood pressure in V1a receptor-deficient (V1aR-/-) mice (Koshimizu T, Nasa Y, Tanoue A, Oikawa R, Kawahara Y, Kiyono Y, Adachi T, Tanaka T, Kuwaki T, Mori T. Proc Natl Acad Sci USA 103: 7807-7812, 2006). In this study, we investigated the role of V1aR in urine concentration, renal function, and the renin-angiotensin system (RAS) using V1aR-/- mice. Urine volume of V1aR-/- mice was greater than that of wild-type mice, particularly when water was loaded, while the glomerular filtration rate (GFR), urinary NaCl excretion, AVP-dependent cAMP generation, V2R, and aquaporin 2 (AQP2) expression in the kidney were lower, indicating that the diminished GFR and V2R-AQP2 system led to impaired urinary concentration in V1aR-/- mice. Since the GFR and V2R-AQP2 system are regulated by RAS, we analyzed renin and angiotensin II in V1aR-/- mice and found that the plasma renin and angiotensin II were decreased. The expression of renin in granule cells was decreased in V1aR-/- mice, which led to a decreased level of plasma renin. In addition, the expression of renin stimulators such as neuronal nitric oxide synthase and cyclooxygenase-2 in macula densa (MD) cells, where V1aR was specifically expressed, was decreased in V1aR-/- mice. These data indicate that AVP regulates body fluid homeostasis and GFR via the V1aR in MD cells by activating RAS and subsequently the V2R-AQP2 system.

Insulin hypersensitivity in mice lacking the V1b vasopressin receptor.

We have reported that [Arg(8)]-vasopressin-stimulated insulin release is blunted in islet cells isolated from V1b receptor-deficient (V1bR(-/-)) mice. In this study, we used V1bR(-/-) mice to examine the physiological role of the V1b receptor in regulating blood glucose levels in vivo, and we found that the fasting plasma glucose, insulin and glucagon levels were lower in V1bR(-/-) mice than in wild-type (V1bR(+/+)) mice. Next, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose and insulin levels during the GTT were lower in V1bR(-/-) mice than in V1bR(+/+) mice. An insulin tolerance test (ITT) revealed that, after insulin administration, plasma glucose levels were lower in V1bR(-/-) mice than in V1bR(+/+) mice. In addition, a hyperinsulinaemic-euglycaemic clamp study showed that the glucose infusion rate was increased in V1bR(-/-) mice, indicating that insulin sensitivity was enhanced at the in vivo level in V1bR(-/-) mice. Furthermore, we found that the V1b receptor was expressed in white adipose tissue and that insulin-stimulated phosphorylation of Akt as an important signaling molecule was increased in adipocytes isolated from V1bR(-/-) mice. Thus, the blockade of the V1b receptor could result, at least in part, in enhanced insulin sensitivity by altering insulin signalling in adipocytes.

Impaired arginine-vasopressin-induced aldosterone release from adrenal gland cells in mice lacking the vasopressin V1A receptor.

We examined aldosterone release in response to stimulation with arginine-vasopressin (AVP) using adrenal gland cells. AVP caused a significant increase in aldosterone release from the dispersed adrenal gland cells of wild-type mice (V1AR+/+) at concentrations from 0.1 microM to 1 microM. In contrast, AVP-induced aldosterone release was impaired in adrenal gland cells from mice lacking the vasopressin V1A receptor (V1AR-/-), while adrenocorticotropic hormone (ACTH)-induced aldosterone release in V1AR-/- mice was not significantly different from that in V1AR+/+ mice. In addition, a vasopressin V1A receptor-selective antagonist 1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone (OPC-21268) potently inhibited AVP-induced aldosterone release. Thus, our study clearly demonstrates that AVP-induced aldosterone release from adrenal gland cells is mediated via the vasopressin V1A receptor in mice.