Rationale The hypothalamic arcuate nucleus (ARC) is considered as a major site for leptin signaling that regulates several physiological processes. Objective To test the hypothesis that leptin receptor in the ARC is required to mediate leptin-induced sympathetic activation. Methods and Results First, we used the ROSA Cre-reporter mice to establish the feasibility of driving Cre expression in the ARC in a controlled manner with bilateral microinjection of adenovirus expressing Cre-recombinase (Ad-Cre). Ad-Cre microinjection into the ARC of ObRflox/flox mice robustly reduced ObR expression and leptin-induced Stat3 activation in the ARC, but not in the adjacent nuclei confirming the efficacy and selectivity of the ARC deletion of ObR. Critically, deletion of ObR in the ARC attenuated brown adipose tissue and renal sympathetic nerve responses to leptin. We also examined if ObR in the ARC is required for the preserved leptin-induced increase in renal sympathetic activity in dietary obesity. We found that deletion of ARC ObR abrogated leptin-induced increases in renal sympathetic discharge and resolved arterial pressure elevation in diet-induced obese ObRflox/flox mice. Conclusions These data demonstrate a critical role for ObR in the ARC in mediating the sympathetic nerve responses to leptin, and in the adverse sympathoexcitatory effects of leptin in obesity.

. (2007) Am. J. Physiol. 293, H2680 -H2692). To understand the underlying mechanisms leading to such cardiac defects, the functional domains of mXin␣ and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXin␣ directly interacts with ␤-catenin. The ␤-catenin-binding site on mXin␣ was mapped to amino acids 535-636, which overlaps with the known actin-binding domains composed of the Xin repeats. The overlapping nature of these domains provides insight into the molecular mechanism for mXin␣ localization and function. Purified recombinant glutathione S-transferase-or His-tagged mXin␣ proteins are capable of binding and bundling actin filaments, as determined by co-sedimentation and electron microscopic studies. The binding to actin was saturated at an approximate stoichiometry of nine actin monomers to one mXin␣. A stronger interaction was observed between mXin␣ C-terminal deletion and actin as compared with the interaction between full-length mXin␣ and actin. Furthermore, force expression of green fluorescent protein fused to an mXin␣ C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXin␣. These results suggest a model whereby the C terminus of mXin␣ may prevent the fulllength molecule from binding to actin, until the ␤-catenin-binding domain is occupied by ␤-catenin. The binding of mXin␣ to ␤-catenin at the adherens junction would then facilitate actin binding. In support of this model, we found that the actin binding and bundling activity of mXin␣ was enhanced in the presence of ␤-catenin.The striated muscle-specific Xin genes encode proteins containing several proline-rich regions, a highly conserved sequence homologous to the Myb-A and Myb-B DNA-binding domain, and a region with 15-28 16-amino acid (aa) 4 repeating units (called the Xin repeats) (1-3). In the mouse, two Xin genes, mXin␣ and mXin␤, exist, whereas only one cXin gene is found in the chick. The expression of both cXin and mXin␣ is regulated by the muscle transcription factor, MEF2C, and the homeodomain transcription factor, Nkx2.5 (1, 2). Similarly, the expression of mXin␤ (also termed myomaxin) is also under the control of MEF2A (4). Treatment of chick embryos with cXin antisense oligonucleotides results in abnormal cardiac morphogenesis and a disruption in cardiac looping, suggesting that Xin plays an essential role in cardiac development (2). Embryonic lethality was expected based on this antisense oligonucleotide experiment in chicks; however, viable and fertile mXin␣ knock-out mice were observed. This viability probably results from functional compensation through the up-regulation of mXin␤ at both message and protein levels (5). Consistent with the compensatory role of mXin␤, we have previously shown that mXin␤, like mXin␣ (2, 6), localizes to the intercalated disc of the adult heart (5). Despite this compensation, the adult mXin␣-deficient mouse hearts are hypertrophied and exhibit c...

SUMMARY The fundamental importance of the hypothalamus in the regulation of autonomic and cardiovascular functions is well established. However, the molecular processes involved are not well understood. Here, we show that the mammalian (or mechanistic) target of rapamycin (mTOR) signaling in the hypothalamus is tied to the activity of the sympathetic nervous system and cardiovascular function. Modulation of mTORC1 signaling caused dramatic changes in sympathetic traffic, blood flow and arterial pressure. Our data also demonstrate the importance of hypothalamic mTORC1 signaling in transducing the sympathetic and cardiovascular actions of leptin. Moreover, we show that PI3K pathway links the leptin receptor to mTORC1 signaling and changes in its activity impacts the sympathetic traffic and arterial pressure. These findings establish mTORC1 activity in the hypothalamus as a key determinant of sympathetic and cardiovascular regulation and suggest that dysregulated hypothalamic mTORC1 activity may influence the development of cardiovascular diseases.

Progress in research and developing therapeutics to prevent diabetic kidney disease (DKD) is limited by a lack of animal models exhibiting progressive kidney disease. Chronic hypertension, a driving factor of disease progression in human patients, is lacking in most available models of diabetes. We hypothesized that superimposition of hypertension on diabetic mouse models would accelerate DKD. To test this possibility, we induced persistent hypertension in three mouse models of type 1 diabetes and two models of type 2 diabetes by adeno-associated virus delivery of renin (ReninAAV). Compared with LacZAAV-treated counterparts, ReninAAV-treated type 1 diabetic Akita/129 mice exhibited a substantial increase in albumin-to-creatinine ratio (ACR) and serum creatinine level and more severe renal lesions. In type 2 models of diabetes (C57BKLS and BTBR mice), compared with LacZAAV, ReninAAV induced significant elevations in ACR and increased the incidence and severity of histopathologic findings, with increased serum creatinine detected only in the ReninAAV-treated mice. The uninephrectomized ReninAAV model was the most progressive model examined and further characterized. In this model, separate treatment of hyperglycemia with rosiglitazone or hypertension with lisinopril partially reduced ACR, consistent with independent contributions of these disorders to renal disease. Microarray analysis and comparison with human DKD showed common pathways affected in human disease and this model. These results identify novel models of progressive DKD that provide researchers with a facile and reliable method to study disease pathogenesis and support the development of therapeutics.

ObjectivesThe autonomic nervous system is critically involved in mediating the control by leptin of many physiological processes. Here, we examined the role of the leptin receptor (LepR) in proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in mediating the effects of leptin on regional sympathetic and parasympathetic nerve activity.MethodsWe analyzed how deletion of the LepR in POMC neurons (POMCCre/LepRfl/fl mice) or AgRP neurons (AgRPCre/LepRfl/fl mice) affects the ability of leptin to increase sympathetic and parasympathetic nerve activity. We also studied mice lacking the catalytic p110α or p110β subunits of phosphatidylinositol-3 kinase (PI3K) in POMC neurons.ResultsLeptin-evoked increase in sympathetic nerve activity subserving thermogenic brown adipose tissue was partially blunted in mice lacking the LepR in either POMC or AgRP neurons. On the other hand, loss of the LepR in AgRP, but not POMC, neurons interfered with leptin-induced sympathetic nerve activation to the inguinal fat depot. The increase in hepatic sympathetic traffic induced by leptin was also reduced in mice lacking the LepR in AgRP, but not POMC, neurons whereas LepR deletion in either AgRP or POMC neurons attenuated the hepatic parasympathetic nerve activation evoked by leptin. Interestingly, the renal, lumbar and splanchnic sympathetic nerve activation caused by leptin were significantly blunted in POMCCre/LepRfl/fl mice, but not in AgRPCre/LepRfl/fl mice. However, loss of the LepR in POMC or AgRP neurons did not interfere with the ability of leptin to increase sympathetic traffic to the adrenal gland. Furthermore, ablation of the p110α, but not the p110β, isoform of PI3K from POMC neurons eliminated the leptin-elicited renal sympathetic nerve activation.Finally, we show trans-synaptic retrograde tracing of both POMC and AgRP neurons from the kidneys.ConclusionsPOMC and AgRP neurons are differentially involved in mediating the effects of leptin on autonomic nerve activity subserving various tissues and organs.