Tammy A. Butterick

Brain orexin promotes obesity resistance

Resistance to obesity is becoming an exception rather than the norm, and understanding mechanisms that lead some to remain lean in spite of an obesigenic environment is critical if we are to find new ways to reverse this trend. Levels of energy intake and physical activity both contribute to body weight management, but it is challenging for most to adopt major long‐term changes in either factor. Physical activity outside of formal exercise, also referred to as activity of daily living, and in stricter form, spontaneous physical activity (SPA), may be an attractive modifiable variable for obesity prevention. In this review, we discuss individual variability in SPA and NEAT (nonexercise thermogenesis, or the energy expended by SPA) and its relationship to obesity resistance. The hypothalamic neuropeptide orexin (hypocretin) may play a key role in regulating SPA and NEAT. We discuss how elevated orexin signaling capacity, in the context of a brain network modulating SPA, may play a major role in defining individual variability in SPA and NEAT. Greater activation of this SPA network leads to a lower propensity for fat mass gain and therefore may be an attractive target for obesity prevention and therapy.

Sleep disorders, obesity, and aging: the role of orexin.

The hypothalamic neuropeptides orexin A and B (hypocretin 1 and 2) are important homeostatic mediators of central control of energy metabolism and maintenance of sleep/wake states. Dysregulation or loss of orexin signaling has been linked to narcolepsy, obesity, and age-related disorders. In this review, we present an overview of our current understanding of orexin function, focusing on sleep disorders, energy balance, and aging, in both rodents and humans. We first discuss animal models used in studies of obesity and sleep, including loss of function using transgenic or viral-mediated approaches, gain of function models using exogenous delivery of orexin receptor agonist, and naturally-occurring models in which orexin responsiveness varies by individual. We next explore rodent models of orexin in aging, presenting evidence that orexin loss contributes to age-related changes in sleep and energy balance. In the next section, we focus on clinical importance of orexin in human obesity, sleep, and aging. We include discussion of orexin loss in narcolepsy and potential importance of orexin in insomnia, correlations between animal and human studies of age-related decline, and evidence for orexin involvement in age-related changes in cognitive performance. Finally, we present a summary of recent studies of orexin in neurodegenerative disease. We conclude that orexin acts as an integrative homeostatic signal influencing numerous brain regions, and that this pivotal role results in potential dysregulation of multiple physiological processes when orexin signaling is disrupted or lost.

Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model

Current data support the idea that hypothalamic neuropeptide orexin A (OxA; hypocretin 1) mediates resistance to high fat diet-induced obesity. We previously demonstrated that OxA elevates spontaneous physical activity (SPA), that rodents with high SPA have higher endogenous orexin sensitivity, and that OxA-induced SPA contributes to obesity resistance in rodents. Recent reports show that OxA can confer neuroprotection against ischemic damage, and may decrease lipid peroxidation. This is noteworthy as independent lines of evidence indicate that diets high in saturated fats can decrease SPA, increase hypothalamic apoptosis, and lead to obesity. Together data suggest OxA may protect against obesity both by inducing SPA and by modulation of anti-apoptotic mechanisms. While OxA effects on SPA are well characterized, little is known about the short- and long-term effects of hypothalamic OxA signaling on intracellular neuronal metabolic status, or the physiological relevance of such signaling to SPA. To address this issue, we evaluated the neuroprotective effects of OxA in a novel immortalized primary embryonic rat hypothalamic cell line. We demonstrate for the first time that OxA increases cell viability during hydrogen peroxide challenge, decreases hydrogen peroxide-induced lipid peroxidative stress, and decreases caspase 3/7 induced apoptosis in an in vitro hypothalamic model. Our data support the hypothesis that OxA may promote obesity resistance both by increasing SPA, and by influencing survival of OxA-responsive hypothalamic neurons. Further identification of the individual mediators of the anti-apoptotic and peroxidative effects of OxA on target neurons could lead to therapies designed to maintain elevated SPA and increase obesity resistance.

Altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning

Altered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated during subsequent ischemia. Fourteen pigs underwent a SWOP protocol with ten 2-minute balloon inflations in the LAD artery, each separated by 2 minutes reperfusion. Twenty-four hours later, mitochondria were isolated from SWOP and SHAM pig hearts and analyzed for uncoupling protein (UCP)-2 content by western blot analysis, proteomic changes by iTRAQ(®) and respiration by an oxygen electrode. In parallel in vivo studies, high-energy nucleotides were obtained by transmural biopsy from anesthetized SWOP and SHAM pigs at baseline and during sustained low-flow ischemia. Compared with SHAM mitochondria, ex vivo SWOP heart tissue demonstrated increased expression of UCP-2, Complex IV (cytochrome c oxidase) and Complex V (ATPase) proteins. In comparison with SHAM pigs during in vivo conditions, transmural energetics in SWOP hearts, as estimated by the free energy of ATP hydrolysis (ΔG(0)), were similar at baseline but had decreased by the end of low-flow ischemia (-57.0 ± 2.1 versus -51.1 ± 1.4 kJ/mol; P < 0.05). In conclusion, within isolated mitochondria from preconditioned SWOP hearts, UCP-2 is increased and in concert with enhanced Complex IV and V proteins, imparts a favorable energetic state during low-flow ischemia. These data support the notion that mitochondrial adaptations that may reduce oxidant damage do not reduce the overall efficiency of energetics during sustained oxygen deprivation.

Amyloid beta-protein1-42 increases cAMP and apolipoprotein E levels which are inhibited by β1 and β2-adrenergic receptor antagonists in mouse primary astrocytes

Amyloid beta-protein (Abeta) increases apolipoprotein E (apoE) levels in astrocytes which could alter lipid trafficking. The mechanism for the Abeta-induced increase in apoE levels is not well understood. It is well established that stimulation of beta-adrenergic receptors (betaARs) increases cAMP levels. Elevation of cAMP levels increases apoE abundance. The current study determined if Abeta(1-42) stimulation of cAMP and apoE levels could be inhibited by betaAR antagonists in astrocytes. We demonstrate that Abeta(1-42) but not the reverse protein Abeta(42-1) or Abeta(1-40) stimulated cAMP formation and this stimulation was inhibited by selective betaAR antagonists in mouse primary cortical astrocytes. Abeta(1-42) significantly increased apoE levels which were significantly inhibited by the betaAR selective antagonists with the greatest inhibition observed with the beta(2) antagonist. Separate lines of evidence have suggested that agonist-induced stimulation of betaARs and increases in apoE abundance may serve a neuroprotective role in astrocytes. Our results indicate a potential interaction between betaARs and apoE which may contribute to reducing Abeta(1-42) neurotoxicity.

Simvastatin Stimulates Production of the Antiapoptotic Protein Bcl-2 via Endothelin-1 and NFATc3 in SH-SY5Y Cells

The use of statins for the prevention or treatment of different neurodegenerative diseases has generated considerable interest albeit with some controversy. Mechanisms of statin-induced neuroprotection are not well understood. Recently, we reported that simvastatin stimulated neuronal gene expression and protein levels of the major antiapoptotic protein Bcl-2 in vivo and in vitro; suppression of Bcl-2 in SH-SY5Y cells reduced simvastatin neuroprotection; effects were independent of cholesterol and other products of the 3-hydroxy-3-methylglutaryl-CoA reductase pathway. Endothelin-1 (ET-1) can increase Bcl-2 abundance via the transcription factor nuclear factor of activated thymocytes (NFATc), and simvastatin was reported to increase ET-1 gene expression. We tested the hypothesis that simvastatin stimulation of Bcl-2 involves up-regulation of ET-1 and binding of NFATc to Bcl-2 promoter sites in SH-SY5Y human neuroblastoma cells. Simvastatin increased both intracellular and secreted ET-1 protein levels. Exogenous ET-1 increased Bcl-2 protein abundance, which was inhibited by ET-1 receptor antagonists. Simvastatin increased translocation of NFATc3 to the nucleus while reducing nuclear NFATc1 and having no effect on NFATc4. Endothelin-1 also increased NFATc3 levels in the nucleus, and this increase was inhibited by ET-1 receptor antagonists. Treatment of cells with simvastatin stimulated binding of NFATc3 to the Bcl-2 promoter. We report novel findings showing that up-regulation of Bcl-2 by simvastatin involves ET-1 and the transcription factor NFATc3. Discovering how statins can selectively alter a specific NFATc isoform that leads to an increase in an antiapoptotic protein will provide a new approach to understanding statin-induced neuroprotection and conditions outside the brain in which apoptosis contributes to pathophysiology.

Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection

Although protection against necrosis has been observed in both hibernating (HIB) and ischemic preconditioned hearts in the second window of protection (SWOP), a comparison of the mitochondrial proteome between the two entities has not been previously performed. Anesthetized swine underwent instrumentation with a fixed constrictor around the LAD artery and were followed for 12 weeks (HIB; N=7). A second group of anesthetized swine underwent ischemic preconditioning by inflating a balloon within the LAD artery 10 times for 2 min, each separated by 2 min reperfusion and were sacrificed 24h later (SWOP; N=7). Myocardial blood flow and high-energy nucleotides were obtained in the LAD region and normalized to remote regions. Post-sacrifice, protein content as measured with iTRAQ was compared in isolated mitochondria from the LAD area of a Sham heart. Basal regional blood flow in the LAD region when normalized to the remote region was 0.86±0.04 in HIB and 1.02±0.02 in SWOP tissue (P<0.05). Despite reduced regional blood flows in HIB hearts, ATP content in the LAD region, when normalized to the remote region was similar in HIB versus SWOP (1.06±0.06 and 1.02±0.05 respectively; NS) as was the transmural phosphocreatine (PCr) to ATP ratio (2.1±0.2 and 2.2±0.2 respectively; NS). Using iTRAQ, 64 common proteins were identified in HIB and SWOP hearts. Compared with SWOP, the relative abundance of mitochondrial proteins involved with electron transport chain (ETC) were reduced in HIB including NADH dehydrogenase, Cytochrome c reductase and oxidase, ATP synthase, and nicotinamide nucleotide transhydrogenase. Within chronically HIB heart tissue with reduced blood flow, the relative abundance of mitochondrial ETC proteins is decreased when compared with SWOP tissue. These data support the concept that HIB heart tissue subjected to chronically reduced blood flow is associated with a down-regulation in the expression of key mitochondrial proteins involved in electron transport.

Orexin: Pathways to obesity resistance?

Obesity has increased in prevalence worldwide, attributed in part to the influences of an obesity-promoting environment and genetic factors. While obesity and overweight increasingly seem to be the norm, there remain individuals who resist obesity. We present here an overview of data supporting the idea that hypothalamic neuropeptide orexin A (OXA; hypocretin 1) may be a key component of brain mechanisms underlying obesity resistance. Prior work with models of obesity and obesity resistance in rodents has shown that increased orexin and/or orexin sensitivity is correlated with elevated spontaneous physical activity (SPA), and that orexin-induced SPA contributes to obesity resistance via increased non-exercise activity thermogenesis (NEAT). However, central hypothalamic orexin signaling mechanisms that regulate SPA remain undefined. Our ongoing studies and work of others support the hypothesis that one such mechanism may be upregulation of a hypoxia-inducible factor 1 alpha (HIF-1α)-dependent pathway, suggesting that orexin may promote obesity resistance both by increasing SPA and by influencing the metabolic state of orexin-responsive hypothalamic neurons. We discuss potential mechanisms based on both animal and in vitro pharmacological studies, in the context of elucidating potential molecular targets for obesity prevention and therapy.

Role of orexin A signaling in dietary palmitic acid-activated microglial cells

Excess dietary saturated fatty acids such as palmitic acid (PA) induce peripheral and hypothalamic inflammation. Hypothalamic inflammation, mediated in part by microglial activation, contributes to metabolic dysregulation. In rodents, high fat diet-induced microglial activation results in nuclear translocation of nuclear factor-kappa B (NFκB), and increased central pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The hypothalamic neuropeptide orexin A (OXA, hypocretin 1) is neuroprotective in brain. In cortex, OXA can also reduce inflammation and neurodegeneration through a microglial-mediated pathway. Whether hypothalamic orexin neuroprotection mechanisms depend upon microglia is unknown. To address this issue, we evaluated effects of OXA and PA on inflammatory response in immortalized murine microglial and hypothalamic neuronal cell lines. We demonstrate for the first time in microglial cells that exposure to PA increases gene expression of orexin-1 receptor but not orexin-2 receptor. Pro-inflammatory markers IL-6, TNF-α, and inducible nitric oxide synthase in microglial cells are increased following PA exposure, but are reduced by pretreatment with OXA. The anti-inflammatory marker arginase-1 is increased by OXA. Finally, we show hypothalamic neurons exposed to conditioned media from PA-challenged microglia have increased cell survival only when microglia were pretreated with OXA. These data support the concept that OXA may act as an immunomodulatory regulator of microglia, reducing pro-inflammatory cytokines and increasing anti-inflammatory factors to promote a favorable neuronal microenvironment.

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.