Shannon M. Harlan

Ablation of the Leptin Receptor in the Hypothalamic Arcuate Nucleus Abrogates Leptin-Induced Sympathetic Activation

Rationale: The hypothalamic arcuate nucleus (ARC) is considered 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 whether 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.

The Intercalated Disc Protein, mXinα, Is Capable of Interacting with β-Catenin and Bundling Actin Filaments

Targeted deletion of mXinα results in cardiac hypertrophy and cardiomyopathy with conduction defects (Gustafson-Wagner, E., Sinn, H. W., Chen, Y.-L., Wang, D.-Z., Reiter, R. S., Lin, J. L.-C., Yang, B., Williamson, R. A., Chen, J. N., Lin, C.-I., and Lin, J. J.-C. (2007) Am. J. Physiol. 293, H2680-H2692). To understand the underlying mechanisms leading to such cardiac defects, the functional domains of mXinα and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXinα directly interacts with β-catenin. The β-catenin-binding site on mXinα 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 mXinα localization and function. Purified recombinant glutathione S-transferase- or His-tagged mXinα 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 mXinα. A stronger interaction was observed between mXinα C-terminal deletion and actin as compared with the interaction between full-length mXinα and actin. Furthermore, force expression of green fluorescent protein fused to an mXinα C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXinα. These results suggest a model whereby the C terminus of mXinα may prevent the full-length molecule from binding to actin, until the β-catenin-binding domain is occupied by β-catenin. The binding of mXinα to β-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 mXinα was enhanced in the presence of β-catenin.

Neuroanatomical determinants of the sympathetic nerve responses evoked by leptin

Leptin is an adipocyte-derived hormone that relays a satiety signal to the brain. The effect of leptin on the sympathetic nervous system is an important aspect in the regulation of energy homeostasis as well as several other physiological functions. The arcuate nucleus of the hypothalamus is considered a major site for the regulation of physiological processes by leptin. However, there is growing recognition that other hypothalamic and extra-hypothalamic brain nuclei are important for leptin regulation of physiological processes including sympathetic nerve traffic. The current review discusses the various hypothalamic and extra-hypothalamic nuclei that have been implicated in leptin-induced increase in regional sympathetic nerve activity. The continuous rise in the prevalence of obesity underscores the importance of understanding the underlying neural mechanisms regulating sympathetic traffic to different tissues to design effective strategies to reverse obesity and associated diseases.

Cardiovascular and sympathetic effects of disrupting tyrosine 985 of the leptin receptor.

Leptin acts in the brain to regulate food intake and energy expenditure. Leptin also increases renal sympathetic nerve activity and arterial pressure. The divergent signaling capacities of the leptin receptor (ObRb) mediate the stimulation of various intracellular pathways that are important for leptin control of physiological processes. We evaluated the cardiovascular and sympathetic consequences of disrupting the signal emanating from tyrosine985 of ObRb. For this, we used LeprL985 (l/l) mice, which carry a loss of function mutation replacing tyrosine985 of ObRb with leucine. Body weight of l/l mice was not significantly different from wild-type controls. In contrast, radiotelemetry measurements revealed that the l/l mice had higher arterial pressure and heart rate as compared with controls. Ganglionic blockade caused a greater arterial pressure fall in the l/l mice relative to controls. In addition, leptin treatment induced a larger increase in arterial pressure and heart rate in the l/l versus wild-type mice. Finally, we compared the response of renal and brown adipose tissue sympathetic nerve activity to intracerebroventricular injection of leptin (2 &mgr;g) between l/l and control mice. Leptin-induced increase in renal sympathetic nerve activity was greater in l/l mice relative to controls. In contrast, the brown adipose tissue sympathetic nerve activity response to leptin was attenuated in the l/l mice relative to controls. These data indicate that selective loss of leptin receptor signaling emanating from tyrosine985 enhances the cardiovascular and renal sympathetic effects of leptin. These findings provide important insight into the molecular mechanisms underlying leptins effects on the sympathetic cardiovascular function and arterial pressure.

PI3K signaling: A key pathway in the control of sympathetic traffic and arterial pressure by leptin

The adipocyte-derived hormone, leptin, is a master regulator of energy homeostasis. Leptin action in the central nervous system also contributes to arterial pressure regulation through its capacity to increase renal sympathetic outflow. The accumulating evidence pointing to a key role for leptin in the adverse sympathetic and cardiovascular consequences of excessive adiposity highlight the importance of understanding the mechanisms underlying the sympathetic and cardiovascular effects of leptin. The ability of the leptin receptor to stimulate various intracellular pathways allows leptin to regulate physiological processes in a specific manner. In this review, we examine the role of the PI3K pathway emanating from the leptin receptor in mediating the sympathetic and arterial pressure effects of leptin. We also discuss the relevance of PI3K signaling for obesity-induced hypertension through its role in mediating selective leptin resistance.

Requirement of TCTG(G/C) Direct Repeats and Overlapping GATA Site for Maintaining the Cardiac‐Specific Expression of Cardiac troponin T in Developing and Adult Mice

The cardiac‐specific −497 bp promoter of rat cardiac troponin T (cTnT) contains two similar modules, D and F, each of which possesses TCTG(G/C) direct repeats and A/T‐rich sites. To identify cis‐elements critical for cardiac specificity, a −249 bp promoter containing only module F and its site‐directed mutations were used to generate transgenic mice. Transgene expression of the −249 bp promoter remained cardiac‐specific, despite low and nonuniform expression. The nonuniform expression pattern of the transgene coincided with differential expression of HMGB1, which appeared to be the predominant form of HMGB family proteins in the heart. The HMGB1 binds to the A/T‐rich/MEF2‐like sites of the cTnT promoter, as determined by chromatin immunoprecipitation assays. Mice carrying the −249 bp promoter with point mutations disrupting the direct repeats expressed transgene at lower levels in the heart and ectopically in the brain. Ectopic expression of transgene was also observed in developing limbs and head. These results suggest an important role for the direct repeat in determining the cardiac specificity. Furthermore, mice carrying a mutant promoter simultaneously disrupting the direct repeats and overlapping GATA site failed to express the transgene in any tissues tested. Therefore, the direct repeat and overlapping GATA site are critical for the expression level and cardiac specificity. The F module controls one level of cardiac specificity. For a uniform and high level of cardiac‐specific expression, the upstream element (−497 to −250 bp) is further required, possibly through the D enhancer module and the combination of Nkx2.5 and GATA sites. Anat Rec, 2008.

Differential contribution of POMC and AgRP neurons to the regulation of regional autonomic nerve activity by leptin

Objectives The 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. Methods We 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. Results Leptin-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. Conclusions POMC and AgRP neurons are differentially involved in mediating the effects of leptin on autonomic nerve activity subserving various tissues and organs.

Characterization of cis-regulatory elements and transcription factor binding: gel mobility shift assay.

To understand how cardiac gene expression is regulated, the identification and characterization of cis-regulatory elements and their trans-acting factors by gel mobility shift assay (GMSA) or gel retardation assay are essential and common steps. In addition to providing a general protocol for GMSA, this chapter describes some applications of this assay to characterize cardiac-specific and ubiquitous trans-acting factors bound to regulatory elements [novel TCTG(G/C) direct repeat and A/T-rich region] of the rat cardiac troponin T promoter. In GMSA, the specificity of the binding of trans-acting factor to labeled DNA probe should be verified by the addition of unlabeled probe in the reaction mixture. The migratory property of DNA-protein complexes formed by protein extracts prepared from different tissues can be compared to determine the tissue specificity of trans-acting factors. GMSA, coupled with specific antibody to trans-acting factor (antibody supershift assay), is used to identify proteins present in the DNA-protein complex. The gel-shift competition assay with an unlabeled probe containing a slightly different sequence is a powerful technique used to assess the sequence specificity and relative binding affinity of a DNA-protein interaction. GMSA with SDS-PAGE fractionated proteins allows for the determination of the apparent molecular mass of bound trans-acting factor.