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1.  Endothelial cell leptin receptor mutant mice have hyperleptinemia and reduced tissue uptake 
Journal of cellular physiology  2013;228(7):1610-1616.
Hyperleptinemia is usually associated with obesity and leptin resistance. Endothelial cell leptin receptor knockout (ELKO) mice without a signaling membrane-bound leptin receptor in endothelia, however, have profound hyperleptinemia without signs of leptin resistance. Leptin mRNA in adipose tissue was unchanged. To test the hypothesis that the ELKO mutation results in delayed degradation and slowed excretion, we determined the kinetics of leptin transfer in groups of ELKO and wildtype mice after intravenous bolus injection of 125I-leptin and the reference substance 131I-albumin. The degradation pattern of 125I-leptin in serum and brain homogenates at different time points between 10-60 min was measured by HPLC and acid precipitation. Although ELKO mice had reduced uptake of 125I-leptin uptake by the brain and several peripheral organs, leptin was more stable in blood and tissue. There was no change in the rate of renal excretion. ELISA showed that serum soluble leptin receptor, known to antagonize leptin transport, had a 400-fold increase, probably contributing to the hyperleptinemia and reduced tissue uptake. Thus, the ELKO mutation unexpectedly increased the stability of leptin but suppressed its tissue uptake. These changes probably contribute to the known partial resistance of the ELKO mice to diet-induced obesity.
doi:10.1002/jcp.24325
PMCID: PMC4091723  PMID: 23359322
Leptin; turnover; endothelia; transport; soluble leptin receptor; ELKO
2.  Protective role of astrocytic leptin signaling against excitotoxicity 
Both pro- and anti-convulsive roles of leptin have been reported, suggesting cell-specific actions of leptin in different models of seizure and epilepsy. The goal of our study was to determine the regulation and function of astrocytic leptin receptors in a mouse model of epilepsy and glutamate-induced cytotoxicity. We show that in pilocarpine-challenged mice developing epilepsy with recurrent seizures after a latent period of 2 weeks, hippocampal leptin receptor (ObR) immunofluorescence was increased at 6 weeks. This was more pronounced in astrocytes than in neurons. In cultured astrocytes, glutamate increased ObRa and ObRb expression, whereas leptin pretreatment attentuated glial cytotoxicity by excess glutamate, reflected by better preserved ATP production. The protective role of astrocytic leptin signaling is further supported by the higher lethality of the astrocyte specific leptin receptor knockout mice in the initial phase of seizure production. Thus, leptin signaling in astrocytes plays a protective role against seizure, and the effects are at least partially mediated by attenuation of glutamate toxicity. Astrocytic leptin signaling, therefore, may be a novel therapeutic target.
doi:10.1007/s12031-012-9924-0
PMCID: PMC3567299  PMID: 23180096
Astrocyte; Leptin; ObR; Epilepsy; Glutamate; Cytotoxicity
3.  Leptin action on non-neuronal cells in the CNS: potential clinical applications 
Leptin, an adipocyte-derived cytokine, crosses the blood-brain barrier to act on many regions of the CNS. It participates in the regulation of energy balance, inflammatory processes, immune regulation, synaptic formation, memory condensation, and neurotrophic activities. This review focuses on the newly identified actions of leptin on astrocytes. We first summarize the distribution of leptin receptors in the brain, with a focus on the hypothalamus where the leptin receptor is known to mediate essential feeding suppression activities, and on the hippocampus, where leptin facilitates memory, reduces neurodegeneration, and plays a dual role in seizures. We will then discuss regulation of the non-neuronal leptin system in obesity. Its relationship with neuronal leptin signaling is illustrated by in vitro assays in primary astrocyte culture and by in vivo studies on mice after pretreatment with a glial metabolic inhibitor or after cell-specific deletion of intracellular signaling leptin receptors. Overall, the glial leptin system shows robust regulation and plays an essential role in obesity. Strategies to manipulate this non-neuronal leptin signaling may have major clinical impact.
doi:10.1111/j.1749-6632.2012.06472.x
PMCID: PMC3407332  PMID: 22530983
leptin; CNS; obesity; astrocytes; blood–brain barrier
4.  Increased Phagocyte-Like NADPH Oxidase and ROS Generation in Type 2 Diabetic ZDF Rat and Human Islets 
Diabetes  2011;60(11):2843-2852.
OBJECTIVE
To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.
RESEARCH DESIGN AND METHODS
Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2′,7′-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.
RESULTS
Levels of phosphorylated p47phox, active Rac1, Nox activity, ROS generation, Jun NH2-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.
CONCLUSIONS
We provide the first in vitro and in vivo evidence in support of an accelerated Rac1–Nox–ROS–JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.
doi:10.2337/db11-0809
PMCID: PMC3198065  PMID: 21911753
5.  Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets 
Biochemical pharmacology  2011;81(8):1016-1027.
Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. secinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. secinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS.
doi:10.1016/j.bcp.2011.01.006
PMCID: PMC3073812  PMID: 21276423
Insulin secretion; pancreatic islet; ARNO; Arf6; Rac1; secinH3
6.  Isoprenylcysteine carboxyl methyltransferase facilitates glucose-induced Rac1 activation, ROS generation and insulin secretion in INS 832/13 β-cells 
Islets  2011;3(2):48-57.
Isoprenylcysteine carboxyl methyltransferase (ICMT) catalyzes the post-translational methylation of C-terminal cysteines of isoprenylated proteins, including small G-proteins and the γ-subunits of heterotrimeric G-proteins. It is widely felt that carboxymethylation promotes efficient membrane association of the methylated proteins and specific protein-protein interactions. In the current study, we tested the hypothesis that ICMT-mediated carboxymethylation of specific proteins (e.g., Rac1) plays a regulatory role in glucose-stimulated insulin secretion (GSIS). Western-blot analysis indicated that ICMT is expressed and predominantly membrane associated in INS 832/13 β-cells. siRNA-mediated knockdown of endogenous expression of ICMT markedly attenuated glucose, but not KCl-induced insulin secretion. These findings were further supported by pharmacological observations, which suggested a marked reduction in glucose-, but not KCl-stimulated insulin secretion by acetyl farnesyl cysteine (AFC), a selective inhibitor of ICMT. In addition, glucose-induced Rac1 activation, a hallmark signaling step involved in glucose-stimulated insulin secretion, was markedly inhibited following pharmacological (AFC) or molecular biological (siRNA-ICMT) inhibition of ICMT. Lastly, we also noticed a marked reduction in glucose-induced acute increase in the generation of reactive oxygen species in INS 832/13 cells pre-treated with AFC or transfected with siRNA-ICMT. Together, these data suggest that ICMT regulates glucose-induced Rac1 activation, generation of reactive oxygen species and insulin secretion in pancreatic β-cells.
doi:10.4161/isl.3.2.15016
PMCID: PMC3092562  PMID: 21346419
Rac1; ROS; pancreatic islet; carboxymethylation and insulin secretion
7.  Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells 
Biochemical pharmacology  2010;80(6):874-883.
The phagocytic NADPH-oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic β-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic β-cells. Incubation of isolated β-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic β-cells.
doi:10.1016/j.bcp.2010.05.006
PMCID: PMC2919057  PMID: 20493824
NADPH Oxidase; Rac1; Tiam1; palmitate; ceramide; oxidative stress; pancreatic β-cells
8.  Leptin action on nonneuronal cells in the CNS: potential clinical applications 
Leptin, an adipocyte-derived cytokine, crosses the blood–brain barrier to act on many regions of the central nervous system (CNS). It participates in the regulation of energy balance, inflammatory processes, immune regulation, synaptic formation, memory condensation, and neurotrophic activities. This review focuses on the newly identified actions of leptin on astrocytes. We first summarize the distribution of leptin receptors in the brain, with a focus on the hypothalamus, where the leptin receptor is known to mediate essential feeding suppression activities, and on the hippocampus, where leptin facilitates memory, reduces neurodegeneration, and plays a dual role in seizures. We will then discuss regulation of the nonneuronal leptin system in obesity. Its relationship with neuronal leptin signaling is illustrated by in vitro assays in primary astrocyte culture and by in vivo studies on mice after pretreatment with a glial metabolic inhibitor or after cell-specific deletion of intracellular signaling leptin receptors. Overall, the glial leptin system shows robust regulation and plays an essential role in obesity. Strategies to manipulate this nonneuronal leptin signaling may have major clinical impact.
doi:10.1111/j.1749-6632.2012.06472.x
PMCID: PMC3407332  PMID: 22530983
leptin; CNS; obesity; astrocytes; blood–brain barrier

Results 1-8 (8)