Chronic spinal cord injury (SCI) results in an accelerated trajectory of several cardiovascular disease (CVD) risk factors and related aging characteristics, however the molecular mechanisms that are activated have not been explored. Adipokines and leptin signaling are known to play a critical role in neuro-endocrine regulation of energy metabolism, and are now implicated in central inflammatory processes associated with CVD. Here, we examine hypothalamic adipokine gene expression and leptin signaling in response to chronic spinal cord injury and with advanced age. We demonstrate significant changes in fasting-induced adipose factor (FIAF), resistin (Rstn), long-form leptin receptor (LepRb) and suppressor of cytokine-3 (SOCS3) gene expression following chronic SCI and with advanced age. LepRb and Jak2/stat3 signaling is significantly decreased and the leptin signaling inhibitor SOCS3 is significantly elevated with chronic SCI and advanced age. In addition, we investigate endoplasmic reticulum (ER) stress and activation of the uncoupled protein response (UPR) as a biological hallmark of leptin resistance. We observe the activation of the ER stress/UPR proteins IRE1, PERK, and eIF2alpha, demonstrating leptin resistance in chronic SCI and with advanced age. These findings provide evidence for adipokine-mediated inflammatory responses and leptin resistance as contributing to neuro-endocrine dysfunction and CVD risk following SCI and with advanced age. Understanding the underlying mechanisms contributing to SCI and age related CVD may provide insight that will help direct specific therapeutic interventions.
Citation: Bigford GE, Bracchi-Ricard VC, Nash MS, Bethea JR (2012) Alterations in Mouse Hypothalamic Adipokine Gene Expression and Leptin Signaling following Chronic Spinal Cord Injury and with Advanced Age. PLoS ONE 7(7): e41073. doi:10.1371/journal.pone.0041073
Editor: Hemachandra Reddy, Oregon Health & Science University, United States of America
Received: January 23, 2012; Accepted: June 18, 2012; Published: July 16, 2012
Copyright: © 2012 Bigford et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was funded by the Miami Project to Cure Paralysis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Traumatic spinal cord injury (SCI) initiates a myriad of primary and secondary mechanisms ,  causing neuronal damage and death, sustained neurological deficits, autonomic and immune dysfunction, and significantly high risk of morbidity and mortality . With major advancements in medical practices  including operative and non-operative treatment strategies ,  and primary rehabilitation , there has been a dramatic increase in the long-term survival rates of persons with SCI , . As such, a consequential shift from mortality in SCI related to acute-phase renal, uroseptic, and respiratory complications, to that of chronically acquired all-cause cardiovascular disease (CVD) has become pervasive –. Several risk factors for CVD and related neuro-endocrine/metabolic disorders, described as the cardiometabolic syndrome, are prevalent in SCI, and include central obesity –, significant dyslipidemia – and depressed plasma HDL-C –, , , as well as impaired fasting glucose and increased prevalence of diabetes mellitus , . Moreover, these risk factors are observed earlier in the lifespan and at increased frequencies in SCI , and current evidence suggests an accelerated trajectory of aging of body systems – and specifically premature aging of the cardiovascular and neuro-endocrine systems . Although substantial evidence supports that CVD risk factors and related nuro-endocrine/metabolic disorders are prevalent in SCI, the biological mechanisms contributing to these comorbidities have yet to be explored.
It is well understood that adipose-derived peptide hormones, described as adipokines, contribute to both peripheral and central neuro-endocrine regulation of energy metabolism , , and that dysregulated expression of several of these factors promote pro-inflammatory responses and metabolic dysfunction  and are implicated in the pathogenesis of obesity, diabetes mellitus and CVD . Increasing evidence support that several adipokines, including fasting-induced adipose factor (FIAF) and resistin (Rstn) are expressed in various regions in the central nervous system (CNS), and exhibit significant changes in mRNA expression following modeled CNS injury , . FIAF and Rstn have been found in key hypothalamic areas responsible for energy balance , however, their signaling and physiological effect are poorly understood, and importantly, their role in central inflammatory and metabolic processes with chronic SCI or with advanced age are not defined.
It has been established that the adipokine leptin governs physiological effects on energy homeostasis through hypothalamic pathways mediated by its cognate long form receptor (LepRb) , –. LepRb initiates Jak2/Stat3 signaling pathways in subpopulations of neurons in the arcuate nucleus (ARC) of the hypothalamus, activating the transcription of the precursor poly-peptide proopiomelanocortin (POMC) which in turn triggers neuro-endocrine pathways associated with metabolic rate, mobilization of energy stores as well as many other growth related processes –. Importantly, prolonged LepRb activation and Jak2/Stat3 signaling induces suppressor of cytokine signaling 3 (SOCS-3) expression mediating feedback inhibition , , which is now understood as a mechanism contributing to acquired leptin resistance and subsequent disruption of neuro-endocrine/metabolic function , .
Hypothalamic inflammation associated with leptin resistance – has been linked in part to endoplasmic reticulum (ER) stress , , where several (ER) stress transducers have been defined. Phosphorylation of ER transmembrane proteins inositol-requiring protein-1 (IRE1), protein kinase RNA (PKR)-like ER kinase (PERK) and its downstream effector eukaryotic translation initiation factor-2 (eIF2α) contribute to transcriptional activation of a complex signaling network, termed the uncoupled protein response (UPR) . Moreover, reduced ER capacity or increased levels of ER stress induce a higher degree of obesity when experimentally challenged with a high fat diet , , . Although these mechanisms have been implicated in metabolic dysfunction, whether or not dysregulated leptin signaling and acquired leptin resistance are induced by chronic SCI or advanced age, has yet to be explored.
Here we investigate central mechanisms that may contribute to over-arching SCI pathophysiology as it relates to CVD risk and neuro-endocrine/metabolic dysfunction, and explore phenotypic similarities with advanced age. We provide evidence that hypothalamic adipokine gene expression is significantly altered chronically following SCI and with advanced age, as well as significant attenuation of hypothalamic LepRb expression and Jak2/Stat3 signaling. In addition, mobilization of the ER stress response and UPR is observed in both conditions. These findings provide evidence for leptin resistance following chronic SCI and with advanced age, which may contribute to neuro-endocrine/metabolic dysfunction, obesity and CVD risk.
Materials and Methods
All animal protocols were approved by the University of Miami Institutional Animal Care and Use Committee (IACUC) and are in accordance with National Research Council guidelines for the care and use of laboratory animals. Young (2–4 months) and aged (13–15 months) C57Bl/6 female mice were used in all experiments described.
Surgeries were performed at the Animal and Surgical Core Facility of the Miami Project to Cure Paralysis according to protocols approved by the IACUC of the University of Miami. Contusion injury was induced with the Infinite Horizon device adapted to the mouse. In brief, mice were anesthetized with an intraperitoneal injection of ketamine (80–100 mg/kg) and xylazine (10 mg/kg). Complete anesthetization was determined by the lack of a stereotypical retraction of the hindpaw in response to a nociceptive stimulus. Mice were then subjected to a laminectomy at vertebrae T9 and the exposed spinal cord was injured at a predetermined impact force of 70 kdynes (severe injury). Sham-operated animals underwent all surgical procedures, including laminectomy, but their spinal cords were not injured. After surgery, animals were housed separately and treated with subcutaneous lactated Ringer's solution to prevent dehydration. Manual bladder expression was performed twice daily. Prophylactic antibiotic gentamicin was administered daily for 7 days to prevent urinary tract infections. Animal tissue was harvested 4-weeks post SCI.
Total ribonucleic acid (RNA) isolation and Quantitative RT-PCR
Total RNA was isolated from mice hypothalamus using the Qiagen RNAeasy mini kit according to the manufacturer's instructions. Two μg of RNA were reverse transcribed using omniscript reverse transcriptase (Qiagen). Real-time PCR was performed with the Rotor-Gene 3000 Real Time Cycler (Corbett Research) on cDNA samples amplified with TAQurate GREEN Real-Time PCR MasterMix (Epicentre Biotechnologies) and primers for FIAF, Rstn, LepRb and SOCS3 (Table 1). Relative expression was calculated by comparison with a standard curve after normalization to β-actin. Between group differences in mRNA expression levels were analyzed using one-way analysis of variance (ANOVA), followed by Tukey post hoc comparison (GraphPad, Prism) and reflect percent change from naïve young (NY) control animals. Single group comparison of sham-operated young and aged animals were analyzed using a two-tailed student's t-test (GraphPad, Prism) and reflect percent change from appropriate naïve control animals. Data are expressed as mean ± SEM. A significance level of p<0.05 was accepted as different from control. n = 5 for each group, and each sample was run in triplicate.
Protein extraction and immunoblot analysis
Mice hypothalami were harvested and homogenized in a Dounce homogenizer with extraction/lysis buffer (w/v) (20 mM Tris–HCl, pH: 7.5, 150 mM NaCl, 1% Triton X-100; 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 2.5 mM pyrophosphate, 1 mM β-glycerophosphate) containing protease and phosphatase inhibitor cocktails (Sigma) and then centrifuged at 15, 300× g for 2 minutes. Lysates were mixed with 2x Laemmli loading buffer. Equal amounts of protein were resolved on 10–20% gradient Tris-HCl Criterion pre-casted gels (Bio-Rad, Hercules, CA), to separate proteins with a wide range of molecular weights, transferred to polyvinylidene fluoride (PVDF) membranes and placed in blocking buffer (0.1% Tween-20, 0.4% I-block in PBS) overnight (38). Membranes were then incubated with primary antibodies followed by the appropriate HRP-conjugated secondary antibody. Visualization of the signal was enhanced by chemiluminescence using a Phototope-HRP detection kit (Cell Signaling). Quantification of bands corresponding to changes in protein levels was made using scanned densitometric analysis and NIH Image Program 1.62f, and normalized to β-Actin or Jak2, Stat3, IRE1, PERK, eIF2α, where appropriate. Between group differences in immunoblots were analyzed using one-way analysis of variance (ANOVA), followed by Tukey post hoc comparison (GraphPad, Prism) and reflect percent change from naïve young (NY) control animals. Single group comparison of sham-operated young and aged animals were analyzed using a two-tailed student's t-test (GraphPad, Prism) and reflect percent change from appropriate naïve control animals. Data are expressed as mean ± SEM. A significance level of p<0.05 was accepted as different from control. n = 8 for each group, and each sample was run in triplicate.
4-weeks post-SCI, animals were anesthetized as described above, then received an intracardial injection of heparin (0.1 cc) and perfused transcardially with physiological saline, followed by 100 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were removed and placed in 4% paraformaldehyde at 4°C for overnight, then transferred to 20% sucrose in 0.1 M PBS until sectioned.
Animals were perfused with 4% paraformaldehyde solution as described above, and brains were processed for cryostat sectioning (Leica SM 2000R sliding microtome). Serial coronal sections (50 μm) (−0.7 mm to −2.4 mm Bregma)  were stored in free-floating cryostat media (30% ethylene glycol, 30% sucrose, 0.1 M PBS, pH 7.4) at −20°C then rinsed with 0.1 M PBS (pH 7.4) Tissue sections were blocked/permeabilized by treatment with 5% normal goat serum (Vector Laboratories Inc., Burlingame, CA, USA) and 0.4% Triton X-100 (Sigma). Sections were incubated for 48 hours at 4°C with either LepRb or NeuN primary antibodies (1∶200). Primary antibody binding was detected with Alexa Fluor secondary antibody conjugates (1∶500, Molecular Probes, Eugene, OR, USA). Controls lacking the primary antibody were run in parallel. Sections were counterstained with DAPI and coverslipped with Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA, USA) for confocal analysis (Olympus, FluoView 1000, scanning confocal microscope).
Rabbit polyclonal anti-Leptin Receptor (long-form, 1∶500, Abbiotec), rabbit polyclonal anti-Jak2P (1∶1000, Cell Signaling), rabbit polyclonal anti-Jak2Total (1∶1000, Cell Signaling), rabbit polyclonal anti-Stat3P (1∶1000, Cell Signaling), rabbit polyconal anti-Stat3Total (1∶1000, Cell Signaling), rabbit polyclonal anti-SOCS3 (1∶500, AbCam), mouse monoclonal anti-β-Actin (1∶2000, Cell Signaling), mouse monoclonal anti-PERKP (1∶1000, Cell Signaling), rabbit polyclonal anti-PERKTotal (1∶1000, Cell Signaling), rabbit polyclonal anti-IRE1P (1∶1000, AbCam), rabbit polyclonal anti-IRE1Total (1∶1000, AbCam), rabbit polyclonal anti-eIF2αP (1∶1000, Cell Signaling), rabbit polyclonal anti-eIF2αTotal (1∶1000, Cell Signaling).
Adipokine, LepRb, and SOCS3 gene expression are significantly altered following SCI and with advanced age
Adipokines modulate central inflammatory responses and metabolic pathways. To investigate whether chronic SCI or advanced age affect central adipokine levels and LepRb signaling intermediates, FIAF, Rstn, LepRb, and SOCS3 gene expression levels were examined using quantitative rt-PCR of hypothalamic extracts from the naïve young (NY), SCI young (SCIY), naïve aged (NO) and SCI aged (SCIO) condition (Figure 1). We observed that FIAF mRNA is significantly increased in the SCI young, naïve aged, and SCI aged animals when compared to naïve young control, and SCI results in significant increases above both the naïve young and naïve aged mice (Figure 1A). Importantly, sham-operated young and aged animals also exhibit significantly increased FIAF mRNA levels when compared to naïve control (Figure S1A), indicating that the differences observed in the SCI condition may not reflect SCI independently. Both naive aged and SCI aged animals show a significant increase in Rstn mRNA levels, when compared to the naïve young control, with no significant difference observed in the SCI young compared to naïve young control (Figure 1B). In addition, the relative amount of LepRb mRNA is significantly reduced in SCI young, naïve aged and SCI aged animals when compared to the naïve young control (Figure 1C), whereas mRNA for the leptin signaling inhibitory intermediate SOCS3 is significantly increased in SCI young, naïve aged and SCI aged animals. (Figure 1D). Sham-operated animals showed no significant difference in Rstn, LepRb, and SOCS3 mRNA level when compared to appropriate naïve control (Figure S1A). These results provide evidence that the hypothalamic adipokine genes and related leptin signaling genes are significantly changed following SCI and with advanced age, which may affect central inflammatory and pathological processes.
(NY), SCI young (SCIY), naïve aged (NO) and SCI aged (SCIO) mice. Quantification of mRNA expression levels show that FIAF is significantly increased in SCIY, NO and SCI O animals compared to NY control. SCIY and SCIO are also significantly increased when compared to NO (A). Rstn mRNA levels are significantly increased in SCIY, NO and SCIO animals compared to NY control. NO and SCIO are also significantly increased when compared to SCIY (B). LepRb mRNA levels are significantly reduced in SCIY, NO and SCIO when compared to NY control (C). SOCS3 mRNA levels are significantly greater in SCIY, NO and SCIO when compared to NY control (D). Statistics are according to data analysis methods described. p≤0.05. n = 5 for each group.
Chronic SCI and advanced age induce a significant decrease in LepRb protein expression, Jak2/Stat3 signaling and concomitant significant increase in SOCS3 protein expression
Since LepRB mediates the anorexigenic effect of leptin in the CNS, we next examined whether chronic SCI or advanced age affected hypothalamic LepRb signaling (Figure 2). Consistent with our mRNA data, we observed that LepRb protein expression is significantly reduced in SCI young, naïve aged, and SCI aged animals when compared to naïve young control. Similarly, Jak2 tyrosine phosphorylation and Stat3 tyrosine phosphorylation are also significantly reduced in SCI young, naïve aged, and SCI aged conditions. In contrast, the expression of the leptin signaling inhibitor SOCS3 is significantly increased with SCI young, naïve aged, and SCI aged animals, when compared to naïve young control, also consistent with our mRNA data. Sham-operated animals showed no significant difference in LepRb, Jak2 (phosphorylated), Stat3 (phosphorylated), and SOCS3 protein expression when compared to appropriate naïve control (Figure S1B). These data suggest that hypothalamic leptin signaling through LepRb is significantly reduced with chronic SCI and advanced age, and may contribute to impaired hypothalamic leptin signaling associated with leptin resistance.
LepRb expression is significantly decreased in SCIY, NO and SCIO animals when compared to the NY control. Similarly, Jak2 phosphorylation and Stat3 phosphorylation are both attenuated in SCIY, NO and SCIO when compared to the NY control. Conversely, the expression level of SOCS3 is significantly elevated in SCIY, NO and SCIO when compared to the NY control. Jak2Total and Stat3Total were used as internal standards. β-Actin was used as a protein loading control. Statistics are according to data analysis methods described. p≤0.05. n = 8 for each group.
Hypothalamic ER stress and activated UPR following SCI and with advanced age
ER stress and the UPR have been shown to play a central role in hypothalamic leptin resistance . Therefore, we analyzed the activation of the ER stress response and the UPR in hypothalami of naïve young control, SCI young, naïve aged, and SCI aged animals (Figure 3). The three cellular stress transducer proteins, IRE1, PERK and eIF2α showed significantly increased phosphorylation in SCI young, naïve aged, and SCI aged animals when compared to naïve young control, indicating increased ER stress and an activated UPR. Sham-operated animals showed no significant difference in IRE1 (phosphorylated), PERK (phosphorylated), and eIF2α (phosphorylated) protein expression when compared to appropriate naïve control (Figure S1C). These data show significantly increased ER stress in the hypothalamus, and provide evidence for central leptin resistance following chronic SCI and with advanced age.
(NY), SCI young (SCIY), naïve aged (NO) and SCI aged (SCIO) mice. IRE1 phosphorylation, PERK phosphorylation and eIF2α phosphorylation are each significantly increased in SCIY, NO and SCIO when compared to the NY control. IRE1Total, PERKTotal, and eIF2αTotal were used as internal standards. Statistics are according to data analysis methods described. p≤0.05. n = 8 for each group.
LepRb localize in subpopulations of cells corresponding to the arcuate nucleus (ARC) of the hypothalamus and are significantly reduced following chronic SCI and with advanced age
It is well established that the arcuate nucleus (ARC) located within the medio-basal hypothalamus contains subpopulations of leptin responsive neurons – that mediate downstream neuro-endocrine signaling pathways responsible for metabolic control. Confocal images (Figure 4) illustrate the regional distribution and cell type expression of LepRb. Here we show that hypothalamic ARC neurons are positively immunostained with LepRb (red) and the neuronal marker NeuN (green) (Row 1) in the naïve young control. Higher magnification images (Row 2) show that LepRb immunostaining is contained within subpopulations of NeuN positive cells, and that LepRb localizes to somatic peripheral membrane regions. In SCI young (Row 3), ARC neurons had substantially less intense LepRb (red) immunoreactivity, also evident at higher magnification (Row 4). Similarly, in naive aged animals (Row 5), LepRb (red) immunoreacivity was substantially reduced when compared to the naïve young control, and is evident at higher magnification (Row 6). Thus, this supports our molecular and biochemical data showing that LepRb expression is significantly reduced following chronic SCI and with advanced age, and additionally, provides evidence that these changes occur in subpopulations of hypothalamic ARC neurons, known to contribute to neuro-endocrine signaling involved in metabolic control.
(NY), SCI young (SCIY) and naïve aged (NO) mice. Mouse brain coronal sections (50 μM; −0.7 mm to −2.4 Bregma) were immunostained with LepRb (Red), the neuronal marker NeuN (Green) and counterstained using DAPI (Blue). In NY mice, brain regions corresponding to hypothalamic ARC (Row 1, Blue) are positively immunostained with LepRb (Row 1, Red) and NeuN (Row 1, Green). Higher magnification (Row 2), confocal images show LepRb (Row 2, Red) immunoreactivity is contained within subpopulations of NeuN (Row 2, green) positive cells and LepRb localizes to peripheral membrane regions on the soma of NeuN positive cells (Row 2, Merged). SCIY mice (Row 3, Row 4) have substantially reduced LepRb (Row 3, 4, Red) immunoreactivity in hypothalamic ARC neurons compared to NY control. Similarly, NO mice (Row 5, 6) display substantially less LepRb (Row 5, Row 6, Red) immunoreactivity in hypothalamic ARC neurons compared to NY control. Scale Bars = 50 μM (Row 1, 3, 5), 10 μM (Row 2,4,6).
Here we show that gene products for the adipokines FIAF, Rstn, and the leptin signaling intermediates LepRb and SOCS3 are present in the mouse hypothalamus, and their expression is significantly altered with either chronic SCI or advanced age. Further, we identified significantly attenuated lepRb protein expression in areas corresponding to the hypothalamic ARC, reduced Jak2/Stat3 signaling, concomitant increases in the leptin signal inhibition protein SOCS3 and activation of the ER stress and UPR proteins IRE1, PERK, and eIF2α. These findings support the idea of adipokine mediated central inflammatory processes and provide evidence for leptin resistance following chronic SCI and with advanced age.
The physical limitations inherent with SCI, as related to movement, musculoskeletal activity and weight bearing contribute to accelerated pathology in cardiovascular and neuro-endocrine health. With these limitations there are subsequent alterations in body composition typified by rapid and long-term declines in muscle mass and increases in central adiposity, which has resulted in the prevalence of obesity with chronic SCI , and predictive for cardiometabolic syndrome and CVD , . CVD has emerged as the leading cause of mortality in chronic SCI , , with greater prevalence of several CVD risk factors compared to the able bodied population , , . Moreover, CVD mortality is significantly greater at earlier ages compared with able-bodied control , supporting SCI pathology as mediating an accelerated trajectory of cardiovascular aging , . A myriad of physiological changes associated with the neurologic injury and impairment contribute to immediate and long-term effects on body systems , . Earlier age related functional declines following chronic SCI have been observed in both the cardiovascular and neuro-endocrine systems. For example, plasma homocysteine – and C-reactive protein (CRP) ,  markers of vascular disease and atherogenesis, are significantly elevated in chronic SCI compared to the able-bodied population, and may contribute to pathological changes in cardiovascular health. Both the extent and neurological level of injury contribute to the development of CVD risk factors in SCI, including dyslipidemia and significant autonomic dysfunction , , , . With this, there is extant imbalance in parasympathetic and sympathetic regulation of cardiovascular control  and may involve cardiovascular nuclei within the CNS, which receive neuronal projections from cortical, mesencephalic, as well as hypothalamic regions –. In this regard, the regulation of energy balance and metabolism via specific hypothalamic nuclei, through both neurological innervation and endocrine function, may be greatly affected following SCI. Elevated serum insulin-like growth factor 1(IGF-1) , , reduced testosterone and human growth hormone – and premature type II Diabetes Mellitus  suggest an hastened decline in neuro-endocrine dysfunction. With growing evidence that centrally-derived adipokine gene expression is sensitive to both peripheral and central inflammatory stimuli –, it is likely that multiple mechanisms contribute to their signaling dysfunction. Our findings extend previous reports that changes in hypothalamic adipokines may contribute to the activation of signal transduction pathways involved in metabolic dysfunction and consequent CVD with chronic SCI and advanced age.
It has been well established that adipokine signal integration function in metabolic homeostasis, and pathological dysfunction in their gene products, signaling, and coordination are implicated in obesity, diabetes and CVD . FIAF is associated with inflammation of the cardiovascular system, particularly endothelial cells  and cardiomyocytes . However, FIAF has also been shown to have beneficial effects on triglyceride lipid metabolism , fatty acid oxidation and lipolysis  as well as plasma glucose levels and glucose tolerance , suggesting an important role in peripheral metabolic homeostasis. Recent evidence has shown FIAF mRNA in the mouse hypothalamus  as well as cultured hypothalamic neurons , and that hypothalamic FIAF participates in central regulation of energy metabolism through AMP kinase-mediated signaling . Importantly, hypothalamic FIAF gene expression is significantly increased in models of brain injury and inflammation , suggesting a signaling role in these pathological processes. Similarly, Rstn has been implicated in a variety of conditions related to the metabolic syndrome, however, the exact mechanism by which Rstn exerts its biological effect are not completely understood. Rstn has been shown to confer glucose intolerance and insulin resistance , , and although no receptor for Rstn has been identified, induction of SOCS3 intracellularly has been suggested as a potential mechanism by which Rstn inhibits insulin signaling . Additionally, Rstn gene expression levels are significantly upregulated in Apo E −/− mice , and are associated with pro-inflammatory markers of atherosclerosis in humans , implicating a role in inflammatory processes involved in atherosclerosis. Importantly, Rstn has previously been identified within the murine ARC, colocalizing with POMC neurons, with marked reduction in leptin deficient mice , suggesting signaling interaction with leptin in the hypothalamus. Our data show for the first time that chronic SCI and advanced age induces a significant increase in hypothalamic FIAF gene expression. It is important to indicate that sham-operated animals also exhibit significant increases in FIAF, and thus it is remiss to attribute the observed increase in FIAF in the SCI condition solely to SCI pathology. Notwithstanding, the SCI administered is more reflective of the clinical condition, mostly associated with spinal column fracture or dislocation , , and in this regard, the results reported may in fact represent an important actuality following SCI. Additionally, we observe significantly greater changes following SCI than with advanced age, suggesting that pathological processes involved in SCI have a greater effect on FIAF than processes associated with advanced age. As well, we report for the first time that chronic SCI and advanced age result in significant increases in Rstn gene expression. Interestingly, there is a significantly greater effect with age when compared to SCI, suggesting that processes associated with age have a greater effect on Rstn that pathological processes associated with SCI. Further experiments identifying specific signaling will be necessary to elucidate both physiological function as well as pathological contribution to metabolic dysfunction.
Leptin effects through hypothalamic-mediated pathways are now well characterized, and dysregulation in signaling and subsequent leptin resistance is implicated in chronic inflammation associated with CVD progression. Leptin activates a complex neural network with component orexigenic and anorexigenic signaling, and includes mesolimbic dopaminergic and brainstem integration involved in feeding, satiety and metabolic homeostasis , . Both humans and mice with genetic loss of function mutations in either leptin or LepRb manifest severe early onset obesity (–, insulin resistance , , dyslipidemia  and other metabolic, neuro-endocrine and immune dysfunctions. Further, evidence has been reviewed  indicating that leptin contributes to the pathogenesis of atherosclerotic vascular disease, with positive correlates between plasma leptin levels and arterial distensibility , intima-media thickness of the common carotid artery , and coronary artery calcification . In fact, in clinically defined coronary atherosclerosis, leptin was determined an independent predictor of future cardiovascular events . As well, leptin has been shown to influence myocardial metabolism and function –, and that hypothalamic leptin signaling may normalize myocardial fatty acid oxidation . These reports demonstrate the importance of leptin-mediated signaling in metabolic regulation and overall cardiovascular health. Our data provide evidence that pathological processes involved with chronic SCI and advanced age result in significant dysfunction in hypothalamic leptin signaling, although we do not observe significantly greater leptin signaling deficits in the SCI aged condition when compared to SCI young or naïve aged animals. Reasonably, this may be due to the fact that with advanced age, leptin signaling is substantially hindered that ancillary SCI has insignificant effects. In fact, as the advanced age phenotype represents a progressively and significantly changed metabolic environment, other mechanism may be more prominent with an accompanying SCI. Here we provide evidence of this, illustrating that FIAF gene expression is increased to a greater extent following SCI when compared to advanced age. We also show significantly reduced hypothalamic LepRb gene product and protein expression with chronic SCI and advanced age in areas localized to hypothalamic ARC. Further, our observations of reduced jak2/stat3 in their phosphorylated (or active) state, and increased SOCS3 expression strengthen that LepRb-mediated signaling is attenuated in chronic SCI and with advanced age.
More recent evidence has shown a direct involvement of ER stress and activated UPR in leptin resistance. The maturation and appropriate folding of proteins occurs through the ER luminal system, and is responsive to programs of cell differentiation, environment, and physiological dynamics . Under pathological conditions, the capacity of the ER is exceeded, initiating concurrent signaling cascades, aimed at reducing protein load in the ER, and transcriptionally activating UPR target genes to participate in protein folding and repair. Specifically, the ER trans-membrane proteins IRE1 and PERK, and the signal transducer eIF2α are activated under ER-stress and initiate the transcription of UPR genes , . Both in vitro and in vivo experiments have demonstrated that ER stress and the activated UPR directly effects leptin signaling and induces leptin resistance. For example, cell lines treated with multiple ER stress inducers, significantly inhibited leptin-induced LepRb, jak2 and stat3 phosphorylation , . In addition, ER stress has been observed as a prominent feature in the hypothalamus of obese mice, and experimentally induced ER stress and the activated UPR result in leptin resistance in lean mice . Conversely, enhancement of ER capacity augments leptin-stimulated LepRB activation , and increases insulin sensitivity and type 2 diabetes in obese mice . These data link obesity, hypothalamic ER stress and the activated UPR, to leptin signaling dysfunction and ultimate leptin resistance, and in this manner, provide biological evidence for leptin resistance in these conditions. We show the induction of ER stress and the activated UPR with chronic SCI and advanced age, although we do not observe significantly greater ER stress and activated UPR in the SCI aged condition when compared to the SCI young and naïve aged condition. In a similar manner to our findings regarding leptin signaling, ER stress and the activated UPR associated with advanced age may reflect a threshold such that accompanying SCI may not incite a significantly greater effect.
We demonstrate the novel findings that chronic SCI and advanced age induce modifications in hypothalamic adipokine genes, dysfunction in LepRb mediated signaling, increased ER stress and activation of the UPR, providing evidence for leptin resistance, which may contribute to metabolic dysfunction and CVD risk in these conditions. Developing an understanding of centrally derived adipokine signaling will help elucidate their physiological role in inflammatory processes, as well as define their contribution to dysfunction under pathological conditions. In particular, leptin signaling involves many central and peripheral processes and tissues, and further experiments will be necessary to define phenotypic changes with chronic SCI and advanced age, and may provide insight into leptin mediated mechanisms involved in metabolic dysfunction and CVD risk and the development of appropriately directed therapeutic countermeasures
Naïve and sham-operated young and aged analysis of hypothalamic adipokine mRNA, leptin signaling intermediates, ER stress and UPR activation. Quantification of mRNA expression levels show that FIAF is significantly increased in sham-operated young (SY) compared to naïve young (NY) control and in sham-operated aged (SO) compared to naïve aged (NO) control (A). Rstn, LepRb and SOCS3 mRNA expression levels are not significantly different in sham-operated young and aged animals when compared to appropriate control (A). LepRb expression, Jak2/Stat3 phosphorylation, and SOCS3 expression are not significantly different in sham-operated young and aged animals when compared to appropriate naïve control (B). IRE1, PERK, and eIF2α phosphorylation is not significantly different in sham-operated young and aged animals when compared to appropriate control (C). Jak2Total, Stat3Total, IRE1Total, PERKTotal, and eIF2αTotal were used as internal standards. β-Actin was used as a protein loading control. Statistics are according to data analysis methods described. p≤0.05. n = 5 for each group.
Conceived and designed the experiments: GEB VCBR. Performed the experiments: GEB VCBR. Analyzed the data: GEB VCBR. Contributed reagents/materials/analysis tools: GEB VCBR JRB MSN. Wrote the paper: GEB VCBR JRB MSN.
- 1. Fehlings LSaM (2001) Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine 26: S2–12.
- 2. Fehlings WJaM (2003) The molecular basis of neural regeneration. Neurosurgery 53: 943–948.
- 3. Cadotte DW, Fehlings MG (2011) Spinal cord injury: a systematic review of current treatment options. Clin Orthop Relat Res 469: 732–741.
- 4. Maayken van den Berg JMC, Jesus de Pedro-Cuesta, Ignacia Mahillo-Fernandez (2010) Survival after Spinal Cord Injury: A systematic Review. Journal of Neurotrauma 27: 1517–1528.
- 5. Ahn H, Fehlings MG (2008) Prevention, identification, and treatment of perioperative spinal cord injury. Neurosurg Focus 25: E15.
- 6. Wahman K, Nash MS, Lewis JE, Seiger A, Levi R (2011) Cardiovascular disease risk and the need for prevention after paraplegia determined by conventional multifactorial risk models: the Stockholm spinal cord injury study. J Rehabil Med 43: 237–242.
- 7. Bauman WA, Spungen AM, Adkins RH, Kemp BJ (1999) Metabolic and endocrine changes in persons aging with spinal cord injury. Assist Technol 11: 88–96.
- 8. DeVivo MJ, Krause JS, Lammertse DP (1999) Recent trends in mortality and causes of death among persons with spinal cord injury. Arch Phys Med Rehabil 80: 1411–1419.
- 9. Garshick E, Kelley A, Cohen SA, Garrison A, Tun CG, et al. (2005) A prospective assessment of mortality in chronic spinal cord injury. Spinal Cord 43: 408–416.
- 10. Myers J, Lee M, Kiratli J (2007) Cardiovascular disease in spinal cord injury: an overview of prevalence, risk, evaluation, and management. Am J Phys Med Rehabil 86: 142–152.
- 11. Nash MS, Mendez AJ (2007) A guideline-driven assessment of need for cardiovascular disease risk intervention in persons with chronic paraplegia. Arch Phys Med Rehabil 88: 751–757.
- 12. Spungen AM, Adkins RH, Stewart CA, Wang J, Pierson RN Jr, et al. (2003) Factors influencing body composition in persons with spinal cord injury: a cross-sectional study. J Appl Physiol 95: 2398–2407.
- 13. Gorgey AS, Gater DR (2011) A preliminary report on the effects of the level of spinal cord injury on the association between central adiposity and metabolic profile. PM R 3: 440–446.
- 14. Liang H, Chen D, Wang Y, Rimmer JH, Braunschweig CL (2007) Different risk factor patterns for metabolic syndrome in men with spinal cord injury compared with able-bodied men despite similar prevalence rates. Arch Phys Med Rehabil 88: 1198–1204.
- 15. Groah SL, Nash MS, Ljungberg IH, Libin A, Hamm LF, et al. (2009) Nutrient intake and body habitus after spinal cord injury: an analysis by sex and level of injury. J Spinal Cord Med 32: 25–33.
- 16. Brenes G, Dearwater S, Shapera R, LaPorte RE, Collins E (1986) High density lipoprotein cholesterol concentrations in physically active and sedentary spinal cord injured patients. Arch Phys Med Rehabil 67: 445–450.
- 17. Bauman WA, Spungen AM, Zhong YG, Rothstein JL, Petry C, et al. (1992) Depressed serum high density lipoprotein cholesterol levels in veterans with spinal cord injury. Paraplegia 30: 697–703.
- 18. Zlotolow SP, Levy E, Bauman WA (1992) The serum lipoprotein profile in veterans with paraplegia: the relationship to nutritional factors and body mass index. J Am Paraplegia Soc 15: 158–162.
- 19. Karlsson AK, Attvall S, Jansson PA, Sullivan L, Lonnroth P (1995) Influence of the sympathetic nervous system on insulin sensitivity and adipose tissue metabolism: a study in spinal cord-injured subjects. Metabolism 44: 52–58.
- 20. Maki KC, Briones ER, Langbein WE, Inman-Felton A, Nemchausky B, et al. (1995) Associations between serum lipids and indicators of adiposity in men with spinal cord injury. Paraplegia 33: 102–109.
- 21. McGlinchey-Berroth R, Morrow L, Ahlquist M, Sarkarati M, Minaker KL (1995) Late-life spinal cord injury and aging with a long term injury: characteristics of two emerging populations. J Spinal Cord Med 18: 183–193.
- 22. Bauman WA, Kahn NN, Grimm DR, Spungen AM (1999) Risk factors for atherogenesis and cardiovascular autonomic function in persons with spinal cord injury. Spinal Cord 37: 601–616.
- 23. Bauman WA, Spungen AM, Raza M, Rothstein J, Zhang RL, et al. (1992) Coronary artery disease: metabolic risk factors and latent disease in individuals with paraplegia. Mt Sinai J Med 59: 163–168.
- 24. Washburn RA, Figoni SF (1999) High density lipoprotein cholesterol in individuals with spinal cord injury: the potential role of physical activity. Spinal Cord 37: 685–695.
- 25. Bauman WA, Spungen AM (2001) Carbohydrate and lipid metabolism in chronic spinal cord injury. J Spinal Cord Med 24: 266–277.
- 26. Nash MS, Lewis JE, Dyson-Hudson TA, Szlachcic Y, Yee F, et al. (2011) Safety, tolerance, and efficacy of extended-release niacin monotherapy for treating dyslipidemia risks in persons with chronic tetraplegia: a randomized multicenter controlled trial. Arch Phys Med Rehabil 92: 399–410.
- 27. Bauman WA, Spungen AM (1994) Disorders of carbohydrate and lipid metabolism in veterans with paraplegia or quadriplegia: a model of premature aging. Metabolism 43: 749–756.
- 28. Kemp BJ (2005) What the rehabilitation professional and the consumer need to know. Phys Med Rehabil Clin N Am 16: 1–18, vii.
- 29. Charlifue S, Jha A, Lammertse D (2010) Aging with spinal cord injury. Phys Med Rehabil Clin N Am 21: 383–402.
- 30. Groah SL, Charlifue S, Tate D, Jensen MP, Molton IR, et al. (2012) Spinal cord injury and aging: challenges and recommendations for future research. Am J Phys Med Rehabil 91: 80–93.
- 31. Hitzig SL, Eng JJ, Miller WC, Sakakibara BM, Team SR (2011) An evidence-based review of aging of the body systems following spinal cord injury. Spinal Cord 49: 684–701.
- 32. Ahima RS, Qi Y, Singhal NS, Jackson MB, Scherer PE (2006) Brain adipocytokine action and metabolic regulation. Diabetes 55: S145–154.
- 33. Ahima RS, Qi Y, Singhal NS (2006) Adipokines that link obesity and diabetes to the hypothalamus. Prog Brain Res 153: 155–174.
- 34. Ouchi N, Parker JL, Lugus JJ, Walsh K (2011) Adipokines in inflammation and metabolic disease. Nat Rev Immunol 11: 85–97.
- 35. Deng Y, Scherer PE (2010) Adipokines as novel biomarkers and regulators of the metabolic syndrome. Ann N Y Acad Sci 1212: E1–E19.
- 36. Brown R, Imran SA, Belsham DD, Ur E, Wilkinson M (2007) Adipokine gene expression in a novel hypothalamic neuronal cell line: resistin-dependent regulation of fasting-induced adipose factor and SOCS-3. Neuroendocrinology 85: 232–241.
- 37. Wilkinson M, Brown R, Imran SA, Ur E (2007) Adipokine gene expression in brain and pituitary gland. Neuroendocrinology 86: 191–209.
- 38. Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, et al. (1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature 379: 632–635.
- 39. Fei H, Okano HJ, Li C, Lee GH, Zhao C, et al. (1997) Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues. Proc Natl Acad Sci U S A 94: 7001–7005.
- 40. Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, Saper CB (1998) Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 395: 535–547.
- 41. Thaler JP, Schwartz MW (2010) Minireview: Inflammation and obesity pathogenesis: the hypothalamus heats up. Endocrinology 151: 4109–4115.
- 42. Kelesidis T, Kelesidis I, Chou S, Mantzoros CS (2010) Narrative review: the role of leptin in human physiology: emerging clinical applications. Ann Intern Med 152: 93–100.
- 43. Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, et al. (1996) Role of leptin in the neuroendocrine response to fasting. Nature 382: 250–252.
- 44. Ahima RS, Saper CB, Flier JS, Elmquist JK (2000) Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 21: 263–307.
- 45. Chan JL, Heist K, DePaoli AM, Veldhuis JD, Mantzoros CS (2003) The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men. J Clin Invest 111: 1409–1421.
- 46. Chan JL, Williams CJ, Raciti P, Blakeman J, Kelesidis T, et al. (2008) Leptin does not mediate short-term fasting-induced changes in growth hormone pulsatility but increases IGF-I in leptin deficiency states. J Clin Endocrinol Metab 93: 2819–2827.
- 47. Howard JK, Flier JS (2006) Attenuation of leptin and insulin signaling by SOCS proteins. Trends Endocrinol Metab 17: 365–371.
- 48. Myers MG, Cowley MA, Munzberg H (2008) Mechanisms of leptin action and leptin resistance. Annu Rev Physiol 70: 537–556.
- 49. Bjorbaek C, Elmquist JK, Frantz JD, Shoelson SE, Flier JS (1998) Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol Cell 1: 619–625.
- 50. Myers MG Jr (2004) Leptin receptor signaling and the regulation of mammalian physiology. Recent Prog Horm Res 59: 287–304.
- 51. Hosoi T, Sasaki M, Miyahara T, Hashimoto C, Matsuo S, et al. (2008) Endoplasmic reticulum stress induces leptin resistance. Mol Pharmacol 74: 1610–1619.
- 52. Zhang X, Zhang G, Zhang H, Karin M, Bai H, et al. (2008) Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity. Cell 135: 61–73.
- 53. Milanski M, Degasperi G, Coope A, Morari J, Denis R, et al. (2009) Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity. J Neurosci 29: 359–370.
- 54. Ozcan L, Ergin AS, Lu A, Chung J, Sarkar S, et al. (2009) Endoplasmic reticulum stress plays a central role in development of leptin resistance. Cell Metab 9: 35–51.
- 55. Cnop M, Foufelle F, Velloso LA (2012) Endoplasmic reticulum stress, obesity and diabetes. Trends Mol Med 18: 59–68.
- 56. Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8: 519–529.
- 57. Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, et al. (2004) Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 306: 457–461.
- 58. Scheuner D, Vander Mierde D, Song B, Flamez D, Creemers JW, et al. (2005) Control of mRNA translation preserves endoplasmic reticulum function in beta cells and maintains glucose homeostasis. Nat Med 11: 757–764.
- 59. Hof PR YW, Bloom FE, Belichenko PB, Cello MR (2000) Comparative cytoarchitectonic atlas of the C57BL/6 and 129/Sv mouse brains. Amsterdam: Elsevier.
- 60. Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, et al. (2001) Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411: 480–484.
- 61. Barsh GS, Schwartz MW (2002) Genetic approaches to studying energy balance: perception and integration. Nat Rev Genet 3: 589–600.
- 62. Grill HJ (2010) Leptin and the systems neuroscience of meal size control. Front Neuroendocrinol 31: 61–78.
- 63. Groah SL, Nash MS, Ward EA, Libin A, Mendez AJ, et al. (2011) Cardiometabolic risk in community-dwelling persons with chronic spinal cord injury. J Cardiopulm Rehabil Prev 31: 73–80.
- 64. Valentine RJ, McAuley E, Vieira VJ, Baynard T, Hu L, et al. (2009) Sex differences in the relationship between obesity, C-reactive protein, physical activity, depression, sleep quality and fatigue in older adults. Brain Behav Immun 23: 643–648.
- 65. Valentine RJ, Vieira VJ, Woods JA, Evans EM (2009) Stronger relationship between central adiposity and C-reactive protein in older women than men. Menopause 16: 84–89.
- 66. Demirel S, Demirel G, Tukek T, Erk O, Yilmaz H (2001) Risk factors for coronary heart disease in patients with spinal cord injury in Turkey. Spinal Cord 39: 134–138.
- 67. Groah SaKM (2010) The state of aging and public health for people with spinal cord injury: Lost in transition? Top Spinal Cord Inj Rehabil 15: 1–10.
- 68. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, et al. (1991) Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 324: 1149–1155.
- 69. Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, et al. (1992) A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA 268: 877–881.
- 70. Bauman WA, Adkins RH, Spungen AM, Waters RL, Kemp B, et al. (2001) Levels of plasma homocysteine in persons with spinal cord injury. J Spinal Cord Med 24: 81–86.
- 71. Liang H CD, Wang Y, Rimmer JH, Braunschweig CL (2007) Different risk factor patterns for metabolic syndrome in men with spinal cord injury compared to able-bodied men despite similar prevalence rates. Arch Phys Med Rehabil 88: 1198–1204.
- 72. Wang TD WY, Hung TS, Su TC, Pan SL, Chen SY (2007) Circulating levels of markers of inflammation and endothelial activation are increased in men with chronic spinal cord injury. J Formos Med Assoc 106: 919–928.
- 73. Groah SL, Weitzenkamp D, Sett P, Soni B, Savic G (2001) The relationship between neurological level of injury and symptomatic cardiovascular disease risk in the aging spinal injured. Spinal Cord 39: 310–317.
- 74. Rosado-Rivera D, Radulovic M, Handrakis JP, Cirnigliaro CM, Jensen AM, et al. (2011) Comparison of 24-hour cardiovascular and autonomic function in paraplegia, tetraplegia, and control groups: implications for cardiovascular risk. J Spinal Cord Med 34: 395–403.
- 75. Teasell RW, Arnold JM, Krassioukov A, Delaney GA (2000) Cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord injury. Arch Phys Med Rehabil 81: 506–516.
- 76. Verberne AJ, Lam W, Owens NC, Sartor D (1997) Supramedullary modulation of sympathetic vasomotor function. Clin Exp Pharmacol Physiol 24: 748–754.
- 77. Verberne AJ, Owens NC (1998) Cortical modulation of the cardiovascular system. Prog Neurobiol 54: 149–168.
- 78. Dampney RA, Coleman MJ, Fontes MA, Hirooka Y, Horiuchi J, et al. (2002) Central mechanisms underlying short- and long-term regulation of the cardiovascular system. Clin Exp Pharmacol Physiol 29: 261–268.
- 79. Grigorean VT, Sandu AM, Popescu M, Iacobini MA, Stoian R, et al. (2009) Cardiac dysfunctions following spinal cord injury. J Med Life 2: 133–145.
- 80. Tsitouras PD, Zhong YG, Spungen AM, Bauman WA (1995) Serum testosterone and growth hormone/insulin-like growth factor-I in adults with spinal cord injury. Horm Metab Res 27: 287–292.
- 81. Wang YH, Huang TS, Lien IN (1992) Hormone changes in men with spinal cord injuries. Am J Phys Med Rehabil 71: 328–332.
- 82. Shetty KR, Sutton CH, Mattson DE, Rudman D (1993) Hyposomatomedinemia in quadriplegic men. Am J Med Sci 305: 95–100.
- 83. Cheville AL, Kirshblum SC (1995) Thyroid hormone changes in chronic spinal cord injury. J Spinal Cord Med 18: 227–232.
- 84. Lavela SL, Weaver FM, Goldstein B, Chen K, Miskevics S, et al. (2006) Diabetes mellitus in individuals with spinal cord injury or disorder. J Spinal Cord Med 29: 387–395.
- 85. Wilkinson M, Wilkinson D, Wiesner G, Morash B, Ur E (2005) Hypothalamic resistin immunoreactivity is reduced by obesity in the mouse: co-localization with alpha-melanostimulating hormone. Neuroendocrinology 81: 19–30.
- 86. Brown R, Thompson HJ, Imran SA, Ur E, Wilkinson M (2008) Traumatic brain injury induces adipokine gene expression in rat brain. Neurosci Lett 432: 73–78.
- 87. Wiesner G, Brown RE, Robertson GS, Imran SA, Ur E, et al. (2006) Increased expression of the adipokine genes resistin and fasting-induced adipose factor in hypoxic/ischaemic mouse brain. Neuroreport 17: 1195–1198.
- 88. Kim HK, Youn BS, Shin MS, Namkoong C, Park KH, et al. (2010) Hypothalamic Angptl4/Fiaf is a novel regulator of food intake and body weight. Diabetes 59: 2772–2780.
- 89. Belanger AJ, Lu H, Date T, Liu LX, Vincent KA, et al. (2002) Hypoxia up-regulates expression of peroxisome proliferator-activated receptor gamma angiopoietin-related gene (PGAR) in cardiomyocytes: role of hypoxia inducible factor 1alpha. J Mol Cell Cardiol 34: 765–774.
- 90. Koster A, Chao YB, Mosior M, Ford A, Gonzalez-DeWhitt PA, et al. (2005) Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism. Endocrinology 146: 4943–4950.
- 91. Mandard S, Zandbergen F, van Straten E, Wahli W, Kuipers F, et al. (2006) The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity. J Biol Chem 281: 934–944.
- 92. Xu A, Lam MC, Chan KW, Wang Y, Zhang J, et al. (2005) Angiopoietin-like protein 4 decreases blood glucose and improves glucose tolerance but induces hyperlipidemia and hepatic steatosis in mice. Proc Natl Acad Sci U S A 102: 6086–6091.
- 93. Wiesner G, Morash BA, Ur E, Wilkinson M (2004) Food restriction regulates adipose-specific cytokines in pituitary gland but not in hypothalamus. J Endocrinol 180: R1–6.
- 94. Brown R, Imran SA, Wilkinson M (2009) Lipopolysaccharide (LPS) stimulates adipokine and socs3 gene expression in mouse brain and pituitary gland in vivo, and in N-1 hypothalamic neurons in vitro. J Neuroimmunol 209: 96–103.
- 95. Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, et al. (2001) The hormone resistin links obesity to diabetes. Nature 409: 307–312.
- 96. Rajala MW, Obici S, Scherer PE, Rossetti L (2003) Adipose-derived resistin and gut-derived resistin-like molecule-beta selectively impair insulin action on glucose production. J Clin Invest 111: 225–230.
- 97. Steppan CM, Wang J, Whiteman EL, Birnbaum MJ, Lazar MA (2005) Activation of SOCS-3 by resistin. Mol Cell Biol 25: 1569–1575.
- 98. Burnett MS, Lee CW, Kinnaird TD, Stabile E, Durrani S, et al. (2005) The potential role of resistin in atherogenesis. Atherosclerosis 182: 241–248.
- 99. Shetty GK, Economides PA, Horton ES, Mantzoros CS, Veves A (2004) Circulating adiponectin and resistin levels in relation to metabolic factors, inflammatory markers, and vascular reactivity in diabetic patients and subjects at risk for diabetes. Diabetes Care 27: 2450–2457.
- 100. Koyanagi I, Iwasaki Y, Hida K, Akino M, Imamura H, et al. (2000) Acute cervical cord injury without fracture or dislocation of the spinal column. J Neurosurg 93: 15–20.
- 101. Sekhon LH, Fehlings MG (2001) Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine (Phila Pa 1976) 26: S2–12.
- 102. Robertson SA, Leinninger GM, Myers MG Jr (2008) Molecular and neural mediators of leptin action. Physiol Behav 94: 637–642.
- 103. Kelesidis T, Daikos G, Boumpas D, Tsiodras S (2011) Does rituximab increase the incidence of infectious complications? A narrative review. Int J Infect Dis 15: e2–16.
- 104. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, et al. (1995) Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540–543.
- 105. White DW, Tartaglia LA (1996) Leptin and OB-R: body weight regulation by a cytokine receptor. Cytokine Growth Factor Rev 7: 303–309.
- 106. Montague CT, Farooqi IS, Whitehead JP, Soos MA, Rau H, et al. (1997) Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 387: 903–908.
- 107. Farooqi IS, Wangensteen T, Collins S, Kimber W, Matarese G, et al. (2007) Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor. N Engl J Med 356: 237–247.
- 108. Oswal A, Yeo G (2010) Leptin and the control of body weight: a review of its diverse central targets, signaling mechanisms, and role in the pathogenesis of obesity. Obesity (Silver Spring) 18: 221–229.
- 109. Friedman JM, Halaas JL (1998) Leptin and the regulation of body weight in mammals. Nature 395: 763–770.
- 110. Harris RB, Zhou J, Youngblood BD, Rybkin, II, Smagin GN, et al. (1998) Effect of repeated stress on body weight and body composition of rats fed low- and high-fat diets. Am J Physiol 275: R1928–1938.
- 111. Farooqi IS, Matarese G, Lord GM, Keogh JM, Lawrence E, et al. (2002) Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest 110: 1093–1103.
- 112. Koerner A, Kratzsch J, Kiess W (2005) Adipocytokines: leptin–the classical, resistin–the controversical, adiponectin–the promising, and more to come. Best Pract Res Clin Endocrinol Metab 19: 525–546.
- 113. Singhal A, Farooqi IS, Cole TJ, O'Rahilly S, Fewtrell M, et al. (2002) Influence of leptin on arterial distensibility: a novel link between obesity and cardiovascular disease? Circulation 106: 1919–1924.
- 114. Ciccone M, Vettor R, Pannacciulli N, Minenna A, Bellacicco M, et al. (2001) Plasma leptin is independently associated with the intima-media thickness of the common carotid artery. Int J Obes Relat Metab Disord 25: 805–810.
- 115. Reilly MP, Iqbal N, Schutta M, Wolfe ML, Scally M, et al. (2004) Plasma leptin levels are associated with coronary atherosclerosis in type 2 diabetes. J Clin Endocrinol Metab 89: 3872–3878.
- 116. Wolk R, Berger P, Lennon RJ, Brilakis ES, Johnson BD, et al. (2004) Plasma leptin and prognosis in patients with established coronary atherosclerosis. J Am Coll Cardiol 44: 1819–1824.
- 117. Nickola MW, Wold LE, Colligan PB, Wang GJ, Samson WK, et al. (2000) Leptin attenuates cardiac contraction in rat ventricular myocytes. Role of NO. Hypertension 36: 501–505.
- 118. Atkinson LL, Fischer MA, Lopaschuk GD (2002) Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis. J Biol Chem 277: 29424–29430.
- 119. Barouch LA, Berkowitz DE, Harrison RW, O'Donnell CP, Hare JM (2003) Disruption of leptin signaling contributes to cardiac hypertrophy independently of body weight in mice. Circulation 108: 754–759.
- 120. Mazumder PK, O'Neill BT, Roberts MW, Buchanan J, Yun UJ, et al. (2004) Impaired cardiac efficiency and increased fatty acid oxidation in insulin-resistant ob/ob mouse hearts. Diabetes 53: 2366–2374.
- 121. Sloan C, Tuinei J, Nemetz K, Frandsen J, Soto J, et al. (2011) Central leptin signaling is required to normalize myocardial fatty acid oxidation rates in caloric-restricted ob/ob mice. Diabetes 60: 1424–1434.
- 122. Yoshida H, Matsui T, Hosokawa N, Kaufman RJ, Nagata K, et al. (2003) A time-dependent phase shift in the mammalian unfolded protein response. Dev Cell 4: 265–271.
- 123. Harding HP, Calfon M, Urano F, Novoa I, Ron D (2002) Transcriptional and translational control in the Mammalian unfolded protein response. Annu Rev Cell Dev Biol 18: 575–599.
- 124. Ozcan U, Yilmaz E, Ozcan L, Furuhashi M, Vaillancourt E, et al. (2006) Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 313: 1137–1140.