ABSTRACT
Mice lacking the RIIß regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) display reduced adiposity and resistance to diet-induced obesity. Here we show that RIIß knockout (KO) mice have enhanced sensitivity to leptin's effects on both feeding and energy metabolism. After administration of a low dose of leptin, the duration of hypothalamic JAK/STAT3 signalling is increased, resulting in enhanced POMC mRNA induction. Consistent with the extended JAK/STAT3 activation, we find that the negative feedback regulator of leptin receptor signalling, Socs3, is inhibited in the hypothalamus of RIIß KO mice. During fasting, RIIß-PKA is activated and this correlates with an increase in CREB phosphorylation. The increase in CREB phosphorylation is absent in the fasted RIIß KO hypothalamus. Selective inhibition of PKA activity in AgRP neurons partially recapitulates the leanness and resistance to diet-induced obesity of RIIß KO mice. Our findings suggest that RIIß-PKA modulates the duration of leptin receptor signalling and therefore the magnitude of the catabolic response to leptin.
Subject(s)
Adiposity/genetics , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/genetics , Hypothalamus/metabolism , Leptin/metabolism , Neurons/metabolism , RNA, Messenger/metabolism , Receptors, Leptin/metabolism , Agouti-Related Protein/metabolism , Animals , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Diet, High-Fat , Feedback, Physiological , Janus Kinases/metabolism , Leptin/pharmacology , Mice , Mice, Knockout , Neuropeptide Y/metabolism , Obesity/genetics , Phosphorylation , Pro-Opiomelanocortin/genetics , Pro-Opiomelanocortin/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/metabolismABSTRACT
Hypothalamic neuronal populations are central regulators of energy homeostasis and reproductive function. However, the ontogeny of these critical hypothalamic neuronal populations is largely unknown. We developed a novel approach to examine the developmental pathways that link specific subtypes of neurons by combining embryonic and adult ribosome-tagging strategies in mice. This new method shows that Pomc-expressing precursors not only differentiate into discrete neuronal populations that mediate energy balance (POMC and AgRP neurons), but also into neurons critical for puberty onset and the regulation of reproductive function (Kiss1 neurons). These results demonstrate a developmental link between nutrient-sensing and reproductive neuropeptide synthesizing neuronal populations and suggest a potential pathway that could link maternal nutrition to reproductive development in the offspring.
Subject(s)
Gene Expression Regulation, Developmental/genetics , Hypothalamus/cytology , Kisspeptins/metabolism , Neurons/metabolism , Pro-Opiomelanocortin/metabolism , Stem Cells/physiology , Agouti-Related Protein/genetics , Agouti-Related Protein/metabolism , Animals , Dependovirus/genetics , Embryo, Mammalian , Genetic Vectors/physiology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunoprecipitation , Kisspeptins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microarray Analysis , Pro-Opiomelanocortin/genetics , RNA, Messenger/metabolism , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolismABSTRACT
PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.
Subject(s)
Aquaporin 2/metabolism , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Diabetes Insipidus/etiology , Animals , Arginine Vasopressin/metabolism , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Hypothalamus/metabolism , Kidney/metabolism , Mice , Mice, Transgenic , Phenotype , Receptors, Vasopressin/metabolism , Water-Electrolyte BalanceABSTRACT
Diphtheria toxin-mediated, acute ablation of hypothalamic neurons expressing agouti-related protein (AgRP) in adult mice leads to anorexia and starvation within 7 d that is caused by hyperactivity of neurons within the parabrachial nucleus (PBN). Because NMDA glutamate receptors are involved in various synaptic plasticity-based behavioral modifications, we hypothesized that modulation of the NR2A and NR2B subunits of the NMDA receptor in PBN neurons could contribute to the anorexia phenotype. We observed by Western blot analyses that ablation of AgRP neurons results in enhanced expression of NR2B along with a modest suppression of NR2A. Interestingly, systemic administration of LiCl in a critical time window before AgRP neuron ablation abolished the anorectic response. LiCl treatment suppressed NR2B levels in the PBN and ameliorated the local Fos induction that is associated with anorexia. This protective role of LiCl on feeding was blunted in vagotomized mice. Chronic infusion of RO25-6981, a selective NR2B inhibitor, into the PBN recapitulated the role of LiCl in maintaining feeding after AgRP neuron ablation. We suggest that the accumulation of NR2B subunits in the PBN contributes to aphagia in response to AgRP neuron ablation and may be involved in other forms of anorexia.
Subject(s)
Appetite/physiology , Neurons/physiology , Pons/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Adjuvants, Immunologic/pharmacology , Agouti-Related Protein/deficiency , Agouti-Related Protein/genetics , Animals , Anorexia/genetics , Anorexia/physiopathology , Anorexia/prevention & control , Appetite/drug effects , Blotting, Western , Body Weight/drug effects , Body Weight/physiology , Deglutition Disorders/genetics , Deglutition Disorders/physiopathology , Deglutition Disorders/prevention & control , Eating/drug effects , Eating/physiology , Lithium Chloride/pharmacology , Male , Mice , Mice, Knockout , Neurons/drug effects , Neurons/metabolism , Phenols , Piperidines/pharmacology , Pons/cytology , Pons/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism , Rhombencephalon/cytology , Rhombencephalon/metabolism , Rhombencephalon/physiology , Time Factors , VagotomyABSTRACT
Agouti lethal yellow (A(y)) mice express agouti ectopically because of a genetic rearrangement at the agouti locus. The agouti peptide is a potent antagonist of the melanocortin 4 receptor (MC4R) expressed in neurons, and this leads to hyperphagia, hypoactivity, and increased fat mass. The MC4R signals through Gs and is thought to stimulate the production of cAMP and activation of downstream cAMP effector molecules such as PKA. Disruption of the RIIbeta regulatory subunit gene of PKA results in release of the active catalytic subunit and an increase in basal PKA activity in cells where RIIbeta is highly expressed. Because RIIbeta is expressed in neurons including those in the hypothalamic nuclei where MC4R is prominent we tested the possibility that the RIIbeta knockout might rescue the body weight phenotypes of the A(y) mice. Disruption of the RIIbeta PKA regulatory subunit gene in mice leads to a 50% reduction in white adipose tissue and resistance to diet-induced obesity and hyperglycemia. The RIIbeta mutation rescued the elevated body weight, hyperphagia, and obesity of A(y) mice. Partial rescue of the A(y) phenotypes was even observed on an RIIbeta heterozygote background. These results suggest that the RIIbeta gene mutation alters adiposity and locomotor activity by modifying PKA signaling pathways downstream of the agouti antagonism of MC4R in the hypothalamus.