REGULATION OF OBESITY AND INSULIN RESISTANCE BY NITRIC OXIDE

Abstract

Obesity is a risk factor for developing type 2 diabetes and cardiovascular disease and has quickly become a world-wide pandemic with few tangible and safe treatment options. While it is generally accepted that the primary cause of obesity is energy imbalance, i.e., the calories consumed are greater than are utilized, understanding how caloric balance is regulated has proven a challenge. Many “distal” causes of obesity, such as the structural environment, occupation, and social influences, are exceedingly difficult to change or manipulate. Hence, molecular processes and pathways more proximal to the origins of obesity—those that directly regulate energy metabolism or caloric intake—appear to be more feasible targets for therapy. In particular, nitric oxide (NO) is emerging as a central regulator of energy metabolism and body composition. NO bioavailability is decreased in animal models of diet-induced obesity and in obese and insulin resistant patients, and increasing NO output has remarkable effects on obesity and insulin resistance. This review discusses the role of NO in regulating adiposity and insulin sensitivity and places its modes of action into context with the known causes and consequences of metabolic disease.

1. The Obesity Epidemic

The recent increase in the prevalence of obesity is cause for alarm. The Centers for Disease Control (CDC) estimates that, from 1962 to 2010, obesity prevalence increased from 13% to 36%. As of 2008, approximately 1.5 billion adults aged 20 years or older were overweight, and 10% were obese [1]; more recent data from the United States indicate that >33% of adults and 17% of children are obese [2]. This has led to a dramatic increase in pre-diabetic states. For example, current estimates indicate that one-third of the population in the United States meets the criteria for pre-diabetes [3, 4], and, in addition to type 2 diabetes (T2D), obesity is closely associated with co-mordities such as cardiovascular disease, hypertension, atherosclerosis, stroke, and cancer [5]. Hence, the current high prevalence of obesity is likely to have a considerable impact on worldwide health. In addition, the economic burden of obesity is substantial and accounts for an estimated $147 billion per year in health care costs [6]. The problem has become so severe that, in 2013, the American Medical Association House of Delegates declared obesity a disease.

The principal cause of obesity is energy imbalance: the calories consumed are greater than that utilized by bodily processes, e.g., breathing, digestion, thermogenesis [7]. Indeed, the average consumption of calories in the US has increased by >200 kcal/d per person, which is partly attributable to the abundance of affordable, widely marketed, energy-dense foods [8-11]. Nevertheless, evidence suggests that the balance between calorie intake and energy expenditure is complex and regulated by many factors. Exposure to increasingly obesogenic environments has been suggested to promote not only overeating, but inactivity as well. For example, the human environment is fraught with both chemical and structural “obesogens.” These include but are not limited to: pollutants that promote adiposity and insulin resistance [12-21]; lack of structural features of the built environment that promote an active lifestyle, such as easy access to parks, sidewalks, and bike paths [22-24]; and the night/day cycles in the natural environment of the individual, which can be altered in those having certain occupations [25-27]. Moreover, the genetic makeup of individuals shows strong associations with the predisposition to become obese [28-30].

Many of these factors influence body composition in an indirect or distal manner, and thus could be considered “distal causes” of obesity (Figure 1). Intervening to address these distal causes is exceedingly difficult. For example, changing the structural environment would likely entail departing from particular types of communities or neighborhoods. Similarly, living under favorable day-night cycles is impossible for workers in some occupations, and changing genetic makeup is currently not an option. Even weight loss via caloric restriction faces difficulties, including an evolution-engendered guard against low fat mass [7, 31] and the propensity of the body to increase caloric efficiency during dieting [32, 33]. The intransigency of these problems has led to a search for causes more proximal to obesity, which may be tangible targets for anti-obesity therapies.

Figure 1. Distal and proximal causes of obesity.

Graphical illustration of the common causes of, or factors that contribute to, obesity: Influencing factors distal to the disease, such as policy as well as structural and chemical “obesogens” of the built and social (cultural) environment, may contribute to the prevalence of obesity. Funding for obesity research, dietary guidelines, physical education policies, and sidewalk standards are examples of potential influences related to Policy, which is most distal to the actual disease. The Built environment, which comprises places created or modified by people—i.e., where individuals work, their transportation systems, and life outside their homes—is another cause distal to obesity. The Social or cultural environment includes those family or cultural influences that affect behavioral activity, occupation (which may involve shift work), and social and media norms, all of which could affect eating habits and physical activity. Lastly, direct mechanisms that control hunger, satiety, energy expenditure, and nutrient absorption are Proximal causes of obesity. Commonly, these proximal causes are more tangible targets for anti-obesity/diabetes therapies compared with distal causes and are commonly regulated by nitric oxide.

2. Metabolic pathways known to regulate obesity

Understanding the mechanisms that promote adiposity and insulin resistance are critical to stem the growing tide of metabolic disease. In particular, the development of therapies for obesity and T2D requires a better understanding of the biochemical pathways that regulate metabolism and body composition. As a first principle, energy balance must be considered to understand how changes in body composition could occur. Any effective obesity treatment must decrease energy intake, increase energy expenditure or both. As discussed later, nitric oxide (NO) plays an important role in many of these proximal causes of obesity, which include:

1. Hunger and satiety: The central nervous system regulates caloric intake and the feeling of satisfaction or fullness after a meal, i.e., satiety. This regulation is dependent on neural and endocrine inputs that can be divided into short- and long-term control systems. Release of cholecystokinin (CCK) in combination with neural signaling in response to gut distension are potent signals of satiety and trigger an end to feeding [34]. The adipose tissue-derived hormone, leptin, is crucial to integrate the melanocortin neuronal circuit of the hypothalamus with the energy stores of the body [34-36]. In addition to leptin, neuropeptide Y (NPY) directly affects feeding behavior, metabolism and body composition [37, 38], and corticotropin-releasing hormone, growth-hormone-releasing hormone, galanin and ghrelin, some of which are expressed in both the stomach and the brain, function in hunger and satiety signaling [39]. The neurotransmitters norepinephrine, dopamine and serotonin are also important in central energy balance [34, 36] and inhibiting their reuptake is the strategy employed by drugs such as sibutramine, which have proven effective but have side effects such as increased blood pressure and heart rate [40]. Other drugs that have been shown effective in decreasing energy intake by suppressing appetite are reviewed elsewhere [7, 41, 42].

2. Nutrient absorption: Targeting nutrient absorption in the gut may be an effective obesity therapy. Signals from the gut released post-prandially are important not only in regulating food intake, but also in digestion and nutrient absorption. Ghrelin and CCK, as well as, peptide YY, glucagon-like peptides 1 and 2, gastric inhibitory peptide and corticotropin-releasing factor function to regulate both signaling and digestion [39, 43, 44]. Inhibition of gastric and pancreatic lipases via orlistat treatment decreases triglyceride hydrolysis and is able to inhibit absorption of ingested fat by ~30% and contributes to a caloric deficit of approximately 200 calories per day [45]. As with neurotransmitter reuptake inhibitors, orlistat promotes weight loss; however, side effects caused discontinuation of the medication in many patients [40].

3. Energy expenditure: The largest contributor to obligatory energy expenditure is the basal metabolic rate (BMR), which is defined as the resting energy expenditure at thermoneutrality in the unfed state [46]. BMR includes cellular turnover, repair and basic functions (e.g., maintenance of ion gradients, transmembrane metabolite transfer), basal synthetic reactions (e.g., RNA, DNA protein synthesis) and mitochondrial proton leak; it also includes obligatory thermogenesis (e.g., digestion and absorption) [7, 46]. Mitochondria are central to the regulation of energy expenditure, and targeting their activity has been a prospect for obesity therapies for decades [47, 48], [49]. Therapies that mimic physiological, anti-obesogenic effects are likely to prove most effective. For example, overexpression of uncoupling protein 1 (UCP1), which increases substrate utilization and electron transport chain turnover, in white adipose tissue [50] or skeletal muscle [51] can prevent diet-induced obesity in mice, suggesting that uncoupling of oxidative phosphorylation in these two organs is sufficient to regulate body composition. Brown fat, which expresses relatively high levels of UCP1, is an exciting target for therapy. Despite the low amounts of brown fat in humans, as little as 50 g of brown fat has been estimated to be capable of utilizing up to 20% of basal caloric needs [52]; and mice with genetically reduced brown adipose tissue (BAT) mass are prone to obesity [53]. The recent discovery that adult humans maintain active depots of BAT [54, 55], in conjunction with the identification of UCP-2 and UCP-3 in the skeletal muscle and other tissues [56, 57], suggests that enhancement of mitochondrial activity may hold promise for combatting obesity.

   The finding that adipocytes in some white adipose tissue depots can be programmed to become similar to brown adipose tissue has further invigorated research into understanding the role of adipose tissue in systemic metabolism. A general idea is to increase mitochondria and UCP1 in white adipose tissue, which could promote a higher BMR. Several molecular targets have been identified, with PPAR-γ-coactivator-1α (PGC-1α) being of critical importance [58]. PGC-1α is a known regulator of energy metabolism and of mitochondrial biogenesis [59] and has been shown to induce many of the characteristic brown fat traits in white adipocytes in vitro [58]. Additionally, recent studies have identified secreted proteins that stimulate brown adipocyte thermogenesis and recruit brown (or beige) adipocytes to white adipose tissue [60] [61].

How can these pathways, as well as others that regulate factors proximal to the cause of obesity, be modulated to stem the tide of the obesity epidemic? Emerging evidence suggests that changes in vascular function could regulate metabolic homeostasis, and many studies show that the gaseous signaling molecule, nitric oxide (NO), may play a pivotal role in regulating systemic metabolism, body composition, and insulin sensitivity. In the sections that follow, we discuss NO, how it is regulated by dietary conditions and obesity, and its known effects on physiology, metabolism and adiposity.

3. Nitric oxide – endogenous formation and general modes of biological action

Nitric oxide and related nitrogen oxides have emerged as critical regulators of cell and tissue function [62]. The potency of NO was perhaps first realized following its inhalation by Sir Humphrey Davy, who nearly died from the self-experiment, and after which he vowed to “never design again…so rash an experiment” [63]. Nearly two centuries later, identification of the cardiovascular processes controlled by NO led to the Nobel Prize in Physiology or Medicine in 1998. Nevertheless, the pleiotropy of NO continues to unfold, and we are now beginning to appreciate the deeper aspects of its impact on metabolism.

Generation of NO

The most common route of NO production is through the action of the nitric oxide synthase (NOS) family of enzymes [62, 64]. NOS enzymes catalyze NADPH- and O₂-dependent oxidation of L-arginine to L-citrulline, producing NO in the process. Such synthesis of NO depends on the availability of cofactors such as FAD, FMN, tetrahydrobiopterin (BH₄), as well as the prosthetic group, heme [65].

The three isoforms of NOS generate NO at different rates [62]. NOS3 (commonly called endothelial NOS or eNOS) is expressed to highest relative abundance in the vascular endothelium, but has also been found in neurons, epithelial cells, and cardiomyocytes [66] as well as adipocytes [67, 68] and hepatocytes [69-71]. It produces relatively low quantities of NO, and its activity is controlled by Ca²⁺ and calmodulin, post-translational modifications [72, 73], and physical forces such as shear stress [74, 75]. NOS1 (frequently called neuronal NOS or nNOS) is also a Ca²⁺/calmodulin-dependent isoform that can be activated by agonists of the N-methyl-D-aspartate (NMDA) receptor [75]. It is expressed mostly in neurons, skeletal muscle, and epithelial cells. Lastly, NOS2 (also termed inducible NOS or iNOS), which has the highest capacity to generate NO, is expressed in multiple cell types in response to inflammatory stimuli [75, 76]. The results of some studies also suggest a mitochondria-localized NOS isoform [77, 78]; however, the specific contribution of this isoform remains unclear. In addition to post-translational modifications and substrate and cofactor availability, NOS activity is regulated by its localization within cells and by interactions with itself and other proteins [75].

In addition to direct production via NOS enzymes, NO can be produced endogenously from the more oxidized nitrogen oxide, nitrite. Reduction of nitrite to NO is increased under acidic and hypoxic conditions, with the reduction occurring enzymatically by heme proteins such as deoxyhemoglobin or deoxymyoglobin [79]. The therapeutic potential of dietary or pharmacological nitrite is supported by multiple studies describing improvements in reperfusion injury following myocardial infarction, in pulmonary hypertension, and injury after organ transplantation [80].

Biochemical properties of NO

NO is a free radical of rather limited biological reactivity. The endogenous half-life of NO has been found to be in the range of 2 ms to > 2 s and appears to depend on the availability of reactants [81]. NO primarily reacts with ferrous (Fe²⁺) iron and with other radical species; such reactions form the basis for nearly all of the biological effects of NO. The highest affinity interactions of NO are with metalloproteins such as soluble guanylate cyclase (sGC), cytochrome c oxidase, and hemoglobin; NO reacts also with non-heme iron. Reaction of NO with Fe²⁺ iron results in the formation of a coordinate bond, which is termed a nitrosyl adduct (i.e., nitrosylation). The presence of free radicals such as superoxide (O₂^.−) [82-85] changes the fate of NO: once reacted, it forms peroxynitrite [82, 84, 85] and can no longer bind to ferrous heme [86]. Peroxynitrite and reactive species derived from it (e.g., nitrogen dioxide) are important in inflammatory responses [85] and can modulate cell signaling [87-90], in part by promoting the oxidation or nitration of a broad range of biomolecules [62].

The NO molecule can also react directly with O₂, which itself is a free radical possessing two unpaired electrons in different π* antibonding orbitals [91]. The reactions of NO with O₂ commonly underlie the mechanisms by which S-nitrosation or S-oxidation of protein side chains occurs. In addition, NO can react directly with thiyl radicals, forming a nitroso covalent bond between NO and the thiol (termed S-nitrosation or S-nitrosylation). Cysteinyl thiols of glutathione and proteins are among the most recognized and studied targets of reactive nitrogen species (RNS). Reaction of RNS with thiols results in the formation of S-nitrosothiols, S-glutathiolated species, and oxidized cysteinyl residues [92]. Such modifications can lead to transient changes in enzyme activity, providing redox switches that can be modulated by addition or removal of the modifications [93].

General physiological roles of NO

NO has multiple biological actions and can regulate physiology acutely or lead to long-term changes in cell function. The pleiotropic roles of NO include the regulation of long-term synaptic transmission, learning, memory, platelet aggregation, leukocyte-endothelial interactions, immune function, and angiogenesis and arteriogenesis (for review, see [94]). However, NO is most well known as a potent regulator of blood flow and was originally termed endothelial-derived relaxing factor (EDRF). The story unfolded from Furchgott and Zawadzki’s initial discovery that endothelial cells control acetylcholine-induced relaxation of smooth muscle [95]. A few years later, NO was identified as the key endothelium-derived molecule promoting vasodilation: NO synthesized by NOS in the endothelium diffuses into the vessel wall where it activates sGC in vascular smooth muscle; this leads to a rise in cyclic GMP (cGMP) and elicits relaxation of the vessel [96-102]. However, it readily became apparent that different isoforms of NOS have different physiological functions. For example, NOS3 and NOS1 were found to have distinct roles in regulating microvascular tone [103]; NOS1 activity in the medulla and hypothalamus is important for systemic regulation of blood pressure [104-107]; and, the nitrergic nerves containing NOS1 are responsible for penile erection [99, 108]. Overall, NO derived from the integration of NOS3 and NOS1 activities play key roles in regulating systemic blood pressure and acutely regulating organ blood flow, whereas NOS2-derived NO species are most well recognized for their impact on pathogen killing and inflammatory processes [82].

Another key function of NO is the regulation of mitochondrial respiration. Acutely, NO inhibits respiration by binding and inhibiting cytochrome c oxidase. Modulation of respiration by NO is dependent on both mitochondrial activity and the O₂ level [109, 110]. In addition, NO directly regulates the binding and release of oxygen with hemoglobin [111] and is able to increase blood flow at sites of very low oxygen concentrations [112]. Thus, a key function of NO is to modulate O₂ gradients in cells and tissues by regulating hemoglobin action and by inhibiting O₂ consumption in respiring mitochondria [81]. Chronic exposure to relatively high levels of NO results in mitochondrial biogenesis [113-115], which could reprogram a cell or tissue to have a higher metabolic capacity.

4. NO bioavailability is diminished in obese and diabetic states

NO bioavailability is decreased in animal models of obesity and diabetes [116, 117] and in obese and diabetic humans [118, 119]. Because its bioavailability is dependent upon the balance between its generation and degradation, diminished levels of NO in obese states may be due to decreased expression of NOS, impairments in NOS activity, or by the reaction of NO with reactive species (e.g., superoxide) (Figure 2). These are discussed in turn below.

Figure 2. Mechanisms for decreased endothelial-derived NO in obesity and diabetes.

Schematic of changes in NOS3 or NO: (A) Decreased NOS3 expression commonly occurs in obese and diabetic states. The mechanisms proposed for diminished expression include TNF-α-mediated destabilization of NOS3 mRNA, which may involve eEF1A1. High levels of NO may regulate NOS3 abundance through cGMP-mediated or via NF-κB-SNO feedback regulatory pathways. A small 27-nt RNA regulates NOS3 expression also, although it is not known whether this mechanism is invoked in obesity or diabetes. (B) Decreased NOS3 activity in obesity and diabetes is largely attributed to insulin resistance, which may be mediated by free fatty acid (FFA)-induced activation of TLR2, TLR4, and NF-κB. In addition, activation of PKCβII may diminish Akt signaling, which normally promotes phosphorylation of NOS3 on Ser1177. Phosphorylation at this site increases NO output by the enzyme. Hyperglycemia may also lead to increased O-GlcNAcylation of NOS3, which decreases Ser1177 phosphorylation and inhibits its activity. In addition, conditions leading to obesity promote upregulation of Cav-1, which is a negative regulator of NOS3, and ceramide accumulation disrupts the NOS3-Akt-HSP90 complex, diminishing activity of the enzyme. (C) NOS3 may also be uncoupled or NO quenched in obese and diabetic states. Diminished levels of substrates and cofactors, such as L-arginine or tetrahydrobiopterin (BH₄), lead to uncoupling of the enzyme, which is commonly associated with the presence of NOS3 monomers rather than dimers and can produce superoxide instead of NO. Endogenous inhibitors of NOS3 such as ADMA are also increased in obese conditions and can promote NOS uncoupling. Elevated production of reactive oxygen species such as superoxide can quench NO and result in its oxidation to highly reactive peroxynitrite, which damages biomolecules and can oxidize BH₄ to BH₂.

NOS expression changes in obesity

A primary mechanism by which NO bioavailability could be decreased is via diminished expression of NOS enzymes (Figure 2A). In particular, lower NOS3 abundance is found in both adipose tissue and skeletal muscle of obese humans and rodents [120-124]. Factors associated with obesity and diabetes including shear stress, lysophosphatidylcholine, oxidized LDL, insulin and lack of exercise can also regulate NOS3 expression [125-129]. In multiple studies, tumor necrosis factor-α (TNFα), which is increased in obesity and implicated in the etiology of insulin resistance [130], was shown to downregulate NOS3 expression and abundance [123, 131-134] by decreasing the stability of NOS3 mRNA [135, 136], effectively shortening its half-life [137]. This destabilization of the NOS3 message was due, at least in part, to upregulation of elongation factor 1-α1 [138].

Acutely, TNF increases NOS3 activity [139], most likely via activation of the PI3KAkt [140] and sphingomyelinase/sphingosine-1-phosphate pathways [141, 142]. Such diametrically opposite acute versus chronic effects of TNF would appear to suggest the potential existence of negative feedback loops that sense high levels of NO, leading to downregulation of NOS3. Indeed, NO donors downregulate NOS3 expression both in vitro and in vivo, which may involve cGMP and/or S-nitros(yl)ation of NF-κB [143, 144]. A small, 27-nt RNA has also been shown to be an effective feedback regulator of NOS3 [145]. Whether such small RNAs or miRNAs that regulate NOS expression are induced with obesity is currently unclear.

Notable changes in the abundance of other NOS isoforms also occur in obesity. The NOS2 enzyme increases in abundance in pancreatic β-cells [146], aorta [147], skeletal muscle [148], liver [149, 150], and adipose tissue [151-153] of obese rodents. In adipose tissue, the majority of NOS2 is derived from infiltrating bone marrow-derived macrophages that display a proinflammatory phenotype [151-153]. Also, high levels of TNFα increase NOS2 in adipocytes, which appears to downregulate UCP-2 [154]; hence, this mechanism could contribute to decreases in white adipose tissue energy expenditure. In the ventromedial hypothalamus, which controls energy intake, diet-induced obesity was associated with lower numbers of NOS1-expressing cells [155]. In the aorta, however, NOS1 was increased in abundance in mice fed a high fat diet. The induction of NOS1 was demonstrated to be due to leptin stimulation [156] and may partially compensate for the loss of NOS3-mediated vasodilatory action that typically occurs in obese, insulin-resistant states.

Changes in NOS3 activity in obesity

Beyond changes in expression, the NO-producing activity of NOS3 is diminished in metabolic disease (Figure 2B). In addition to the required substrates, calcium, and cofactors, the activity of NOS3 is regulated by protein-protein interactions and by several post-translational modifications [94, 157, 158]. A high fat diet was shown to upregulate caveolin-1, a negative regulator of NOS3 [159, 160], in the aorta of obese rats [161]. Furthermore, ceramide (which is increased in obesity [162]) promotes disruption of the eNOS-Akt complex from HSP90 [163], which normally increases NOS3 activity by promoting displacement of caveolin-1 from NOS3 [164].

Conditions of obesity have profound effects on NOS3 phosphorylation. In particular, NOS3 phosphorylation at serine 1177 (S1177; S1176 in mice), which is critical for increasing NO output from the enzyme [165], is diminished in mice by nutrient excess [166-169] or high fat feeding [117, 122, 170, 171]; studies in obese rats [172-174] and pigs [175] show similar results. This NOS3 phosphorylation site can be regulated by Akt [176], which is activated by insulin [177]. Insulin stimulation of the Akt-NOS3 pathway could thus be important for regulating post-prandial blood flow and nutrient disposition to peripheral tissues. Indeed, insulin resistance in the endothelium is sufficient to diminish NO bioavailability and promote endothelial dysfunction [178], and impaired NOS3 phosphorylation due to insulin resistance was shown to be responsible for diminished glucose uptake in the skeletal muscle of mice subjected to nutrient excess [179].

The reasons for diminished phosphorylation of NOS3 under conditions of nutrient excess and obesity could be due to fatty acid (e.g., palmitate)-mediated induction of insulin resistance [117]. Elevated free fatty acids lower NO bioavailability in cultured cells [180], isolated arteries [181], animal models [182] and humans [183, 184] and this is likely due to decreased phosphoinositide 3-kinase (PI3K)-Akt-mediated activation of NOS3 [185]. Insulin resistance due to FFAs may be engendered by activation of Toll-like receptor 4 (TLR4) and NF-κB [170, 180] or Toll-like receptor 2 (TLR2) [186]. Other nutrient conditions inherent to diabetes may also be responsible for loss of S1177-NOS3 phosphorylation. For example, hyperglycemia causes O-linked N-acetylglucosamine (O-GlcNAc) modification of NOS3, which diminishes its activity [187]. Other mechanisms posited for diminished NOS3 phosphorylation in the context of obesity include a fatty acid-mediated, yet Akt-independent impairment of NOS3 phosphorylation [171], and PKCβII-mediated diminishment in Akt and NOS3 responsiveness to insulin [172, 174].

Interestingly, insulin-sensitizing therapeutic agents have robust effects on both NOS3 and NOS2. Several glitazone derivatives (also known as thiazolidinediones) used to treat type 2 diabetes increase NOS3 activity or expression in cultured endothelial cells [188-191]. Furthermore, the anti-diabetes drug metformin was shown to stimulate NOS3 activity in vitro and in vivo in an AMP kinase-dependent and HSP90-mediated manner [192]. Conversely, insulin-sensitizing PPARγ agonists robustly repress NOS2 expression in multiple cell and tissue types [193-198].

Uncoupling of NOS and quenching of NO in metabolic disease

The ability of NOS to produce NO is also dependent on its proper coupling, which is regulated by multiple cofactors, the ability of the NOS enzyme to remain in the dimerized form [199, 200],and post-translational modifications [94, 201-203] (Figure 2C). In particular, the cofactor BH₄ is critical to NOS activity, and it has been termed a ‘redox sensor’ because elevations in reactive oxygen and nitrogen species can result in its depletion [204]. Furthermore, BH₄ has been suggested to reflect the overall ‘health’ of the endothelium [205]. Obese and diabetic states in rodents and human cells are associated with decreased BH₄ and elevated levels of its oxidized form, BH₂ [205-209]. This is important because deficiency in BH₄ or elevations in BH₂ can uncouple NOS, which results in superoxide production from the enzyme and increases peroxynitrite generation [201]. Hence, deficiency of BH₄ is thought to be a major regulator of vascular dysfunction that occurs during obesity and in diabetic states. Indeed, the ratio of BH₄ to BH₂ is critical in preventing glucose-induced NOS3 uncoupling [210] and replenishment of BH₄ pools via either providing sepiapterin or by inducing pathways that increase biopterin synthesis or maintain BH₄ in its reduced state have proven effective in multiple pathological scenarios (for review, see [201, 202, 211-213]). Uncoupling of NOS does not appear to be a factor unique to NOS3, however, as NOS1 was shown to be uncoupled in penile arteries of obese rats, leading to nitrergic dysfunction, which was corrected by increasing BH₄ levels [214].

Peroxynitrite may be especially critical in promoting NOS uncoupling. 3-nitrotyrosine (3-NT) is a typical ‘footprint’ post-translational modification that helps identify the sites at which NOS3 uncoupling might have occurred, and it is worth noting that this modification is observed in abundance in the context of obesity and diabetes (e.g., [122, 206, 215, 216]). Patients with diabetes had diminished flow-mediated dilation of coronary arterioles and increased 3-NT protein adducts that colocalized with caveolae, demonstrating a dysfunction of the endothelium associated with elevated peroxynitrite production [217]. Interestingly, endothelial dysfunction in the diabetic patients was rescued by sepiapterin supplementation [217], inferring that peroxynitrite may disrupt NOS3 function not only by caveolar disruption, but by depleting BH₄. This would be consistent with multiple studies showing that elevated levels of reactive species (in addition to peroxynitrite, such as superoxide produced from NADPH oxidase) promote NOS3 uncoupling [218-222]. Nevertheless, the specific contribution of peroxynitrite and other reactive species to endothelial function is still unclear, as other studies suggest that rather than uncoupling NOS3, superoxide from NADPH oxidase activates the enzyme; hence, inhibited NOS3 function perceived under conditions of oxidative stress could be due in part to the quenching of NO and not to uncoupling of the enzyme per se [223]. While this would be consistent with the near diffusion-limited reaction rate of NO with superoxide (which is reported to be as high as 1.9 × 10¹⁰ M⁻¹ s⁻¹) [224], the evidence for a deleterious role of uncoupled NOS should not be underestimated, and multiple other factors beyond BH₄ depletion, such as asymmetric dimethyl arginine (ADMA), insufficient L-arginine levels or glutathio(ny)lation of the NOS3 enzyme, can promote NOS3 uncoupling and endothelial dysfunction [94, 225-228]. That levels of ADMA are positively correlated with insulin resistance and diabetes and that arginine supplementation overcomes this competitive inhibition [229] further suggests that losses in NOS3 coupling or activity are major contributors to the development of metabolic diseases associated with obesity.

Despite these findings, it is unclear whether obesity itself decreases NO availability. The fact that obesity in humans is associated with decreased blood flow in response to methacholine [230], bradykinin [231, 232], substance P and acetylcholine [232], shear stress [233], and insulin [234, 235] appears to suggest that the obese condition is somehow linked causally with diminished vascular NO bioavailability; a multitude of studies showing similar results lends support to this hypothesis [120, 236-247]. However, the question remains: Is loss of NO production somehow due to excess adiposity, or is its etiology derived from those conditions commonly associated with obesity? Interestingly, endothelial dysfunction was found to occur in morbidly obese humans only when insulin resistance was present [248]. And, severely obese humans, in the absence of insulin resistance, showed better flow-mediated dilation compared with normal and obese insulin-sensitive subjects [249]. Furthermore, capillary recruitment has been shown to be higher in overweight compared with lean individuals [250]. This suggests that the maintenance of a metabolically benign form of obesity is possible and that either insulin resistance or conditions directly linked with the insulin resistant phenotype (e.g., dyslipidemia, inflammation, hyperglycemia) are to blame for loss of NO bioavailability during obesity. Collectively, these findings bring forth multiple questions: What determines how metabolically benign versus harmful forms of obesity evolve?; How does NO affect obesity and insulin resistance?; and, What is the relevance of changes in NOS isoform abundance, some of which go in diametrically opposite directions (e.g., NOS3 vs. NOS2), in the development of metabolic disease?

5. Regulation of obesity and insulin resistance by NO

Both pharmacological studies and gain-of-function and loss-of-function studies have helped elucidate critical roles for NO in regulating obesity and insulin resistance. To date, supplementation with the NOS substrate, L-arginine, and inhibition of NOSs have been the most common pharmacological approaches used to determine how NO regulates body composition and insulin sensitivity. Genetic approaches, utilizing mice in which components integral to the synthesis of nitric oxide have been deleted or overexpressed, have further led to the development of a model by which NO regulates systemic metabolism. The model is complex and involves nearly all aspects thought to be important in regulating metabolic homeostasis.

Lessons from pharmacological interventions

Early pharmacological studies, using primarily L-arginine and NOS inhibitors, showed that NO is a potent regulator of both energy intake and expenditure. Interestingly, both L-arginine and inhibitors of NOS prevent obesity and insulin resistance, albeit by different mechanisms.

NO increases food intake

In rodents, L-arginine was shown to increase, and NOS inhibitors to decrease, food intake [155, 251-254]. These effects were due to NO activity in the brain, impinging on the leptin and serotonergic systems that regulate hunger. Leptin, given intracranially, was found to diminish diencephalic NOS activity and decrease food intake and body weight gain, and intracranial co-administration of L-arginine antagonized this effect [255]. Furthermore, intracerebroventricular injection of L-arginine, likely through stimulation of NOS activity, inhibited serotonin-induced anorexia caused by IL-1β [256]. Studies with NOS inhibitors have further solidified our understanding of the central effects of NO on hunger. Systemic administration of the NOS inhibitor, NG-nitro-L-arginine, reduced food intake in obese rats and increased serotonin metabolism in the cortex, diencephalon, and medulla pons, thereby implicating the central serotoninergic system in mediating the anorexic effect of NOS inhibitors [257]. Other NOS inhibitors, such as L-NAME, promote weight loss and diminish food intake in ob/ob and db/db mice [253] and obese rats [258] as well as reduce adiposity and improve insulin sensitivity in high fat-fed mouse models [259]. Interestingly, intracerebroventricular administration of NG-monomethyl-L-arginine (LNMMA) was shown also to regulate insulin secretion and peripheral insulin sensitivity [260], suggesting that centrally derived NO has effects that extend to distal nodes of systemic metabolism. It is also possible that this effect contributes to the hyperphagic effects of NO, as insulin is well known to regulate hunger and satiety [261-265]. Taken together with numerous other studies demonstrating a role for NO in the regulation of hunger [266-270], it would appear that NO produced in the brain antagonizes anorectic signals and stimulates food intake.

NO increases energy expenditure and regulates glucose and lipid metabolism

Ostensibly, this might imply that by promoting food intake, increased levels of NO, e.g., that elicited by supplementation with L-arginine, should increase adiposity and insulin resistance. However, human studies have repeatedly shown that L-arginine supplementation has favorable effects on body composition and insulin sensitivity [271-277]. Results from animal studies are in general agreement: L-arginine has been shown to have multimodal effects characterized by decreased fat mass, increased muscle mass, and improved insulin sensitivity. Despite promoting hyperphagia, L-arginine feeding reduced white adipose tissue mass, improved insulin sensitivity, and increased energy expenditure in mice [278]. In rats, not only has dietary L-arginine supplementation been shown to reduce fat mass, but it appears to increase skeletal muscle and brown fat mass and reduce serum concentrations of glucose, triglycerides, free fatty acids, homocysteine, dimethylarginines, and leptin as well [279, 280]. Similar salubrious systemic effects of L-arginine were demonstrated in pigs [281]. Overall, the collective data suggest that L-arginine, and by inference, NO, has the capacity to reduce fat mass by increasing mitochondrial biogenesis, regulating brown adipose tissue signaling, and increasing the expression of genes that promote oxidation of energy substrates [282].

Chronic treatment with sildenafil, which prevents the degradation of cGMP and is commonly prescribed to improve erectile function, improved insulin action and diminished obesity in high fat-fed mice [283]. Shorter durations of sildenafil treatment were shown to promote “browning” of white adipose tissue [284]. While there are no reports of regulation of obesity in humans by type 5-phosphodiesterase inhibitors such as sildenafil, they have been shown to increase mitochondrial biogenesis in human adipose tissue ex vivo [285], suggesting at least the potential to promote energy expenditure.

Other compounds that function in the NO pathway lend additional support to a role for NO in regulating insulin sensitivity. Restoration of NOS3 phosphorylation in endothelial-specific insulin receptor substrate 2 (IRS-2) knockout mice using beraprost (a stable prostaglandin analog) was sufficient to rescue capillary recruitment and to promote adequate insulin and glucose delivery to skeletal muscle [179]. Insulin, L-arginine, and sodium nitroprusside, by promoting S-nitro(sy)lation of key proteins, were shown to be particularly critical for regulating vascular endothelial insulin uptake and its transendothelial transport [286]. Hence, NO derived from NOS3 appears to be pivotal for regulating systemic glucose metabolism and insulin delivery to peripheral tissues.

As has been a theme with NO, its effects depend on its site of generation, concentration, and duration of application. Particularly interesting are its modes of action in the liver, skeletal muscle, and pancreas. Although chronic treatment with NOS inhibitors promote weight loss and insulin sensitivity in animal models [253, 258, 259], acute applications of inhibitors of NOS cause systemic insulin resistance [287]. This, in part, is mediated by actions in the liver, which can regulate systemic responses to insulin [288]. Administration of BH₄, which is known to be oxidized to BH₂ in the diabetic state [289-291] and plays an important role in regulating coupled NOS3 activity (vide supra), to STZ-induced diabetic mice lowered fasting blood glucose levels in an NOS3-dependent manner and improved glucose tolerance and insulin sensitivity in ob/ob mice [292]. This metabolic improvement was at least partially due to NOS3-mediated activation of AMPK in the liver, which suppressed hepatic gluconeogenesis [292]. Hence, the activity of NOS3 uncoupling in liver may be important for regulating systemic glucose metabolism.

Other studies also demonstrate an important role for liver NOS3 and nitrogen oxides. For example, intraportal administration of NOS inhibitors was shown to cause insulin resistance, which was rescued by intraportal delivery of the NO and superoxide donor, SIN-1 [293, 294]. Interestingly, when liver glutathione was first depleted by buthionine sulfoximine, the effects could not be rescued by sodium nitroprusside or SIN-1 [294]. These results suggest that nitrosated glutathione (GSNO) might be important in maximizing liver-mediated systemic responses to insulin. That intraportal delivery of glutathione methyl ester and SIN-1 enhances insulin sensitivity in rats would appear to support this hypothesis [295].

The NO-HISS connection?

This adds an additional question: How does NO (and its oxidation products) in the liver mediate systemic responses to insulin? The answer has been suggested to lie in a hormone called ‘hepatic insulin-sensitizing substance (HISS)’. This substance, for which there are only suggestive candidates (e.g., bone morphogenetic protein-9 [296]), has been suggested to account for 55% of the glucose disposal effect of insulin. Briefly, it is posited that post-prandial elevations in insulin result in release of a hormone, i.e., HISS, from the liver that acts on skeletal muscle to promote glucose uptake [297, 298]. Intriguingly, one study suggests that HISS, not insulin action, regulates the peripheral vasodilation generally attributed to insulin [299]. Atropine or hepatic surgical denervation inhibited the peripheral vascular actions of insulin, allegedly by blocking HISS release, and intraportal delivery of acetylcholine, which increases NOS activity, restored HISS release and insulin-mediated vasodilation [299]. This is consistent with original studies showing that insulin-mediated vasodilation is dependent on NO [300, 301] and that insulin-mediated skeletal muscle vasodilation contributes to insulin sensitivity in humans [302]; and, combined with other studies suggesting a role for NO in promoting release of HISS [303-305], this suggests that the putative hormone could be an NO-regulated, liver-produced, endocrine mediator of classical EDRF important for glucose disposal. What remains to be elucidated (in addition to the identity of HISS) is how this distally engendered mode of vasoregulation integrates physiologically (and pathologically) with the known local effects of insulin and NO in the vasculature [176-179].

Pancreatic effects of NO

Extremely important for maintaining metabolic homeostasis, the pancreas also utilizes NO to regulate its function. Briefly, the pancreas is comprised of two types of glands: (1) exocrine glands, which secrete the bicarbonate and digestive enzymes needed to neutralize the acidic gastric contents entering the small intestine and to complete digestion of food, respectively; and (2) endocrine glands, i.e., the islets of Langerhans, which contain several types of secretory cells, including α cells, β cells, δ cells, and F cells. Each of these cells secretes multiple proteins, such as insulin (β cells), glucagon (α cells), and somatostatin (δ cells). NO has been shown to affect both exocrine and endocrine functions of the pancreas. The effects of NO on the exocrine actions of the pancreas can be found in [306, 307].

With respect to insulin release, it appears that NO stimulates early, glucose-induced insulin release, while it is responsible for cytokine (e.g., IL-1β)-mediated inhibition of insulin secretion. This dual role of NO in regulating insulin secretion has been the subject of controversy (e.g., [308]), which might be based, in part, on the mechanistic complexity regulating pancreatic insulin secretion, and is likely compounded by the multiple actions of NO. The inhibitory actions of NO on insulin release appear to be due to NOS2-derived NO, which is implicated in the destruction of islet cells in type 1 diabetes [309, 310]. The mechanisms regulating the insulin-stimulating effects of NO [311-314] have been more difficult to elucidate. However, NO is suggested to stimulate islet cell insulin secretion by inducing calcium release from mitochondria [315], which may be due to NO-mediated inhibition of respiration and mitochondrial depolarization. Nevertheless, the stimulatory effects of NO on insulin secretion are relatively subtle [314], which might explain why other studies suggest that NO is not involved in the initiation of insulin secretion from pancreatic islets [316, 317].

Lessons from human studies and genetic interventions

Considerable data has accumulated suggesting an association between genetic polymorphisms in NOS isoforms and insulin resistance. Most notably, several studies have associated a T(−786)C variant of the NOS3 gene with insulin resistance [318-320]. Several other genetic variants in the NOS3 locus are associated with T2D [321], susceptibility for insulin resistance, hypertriglyceridemia, and low HDL [322], or worsened endothelial function in individuals prone to T2D [323]. Polymorphisms in the NOS2 gene have been associated with higher plasma glucose and elevated waist/hip ratios [324], and variants in the NOS2 gene promoter were found to be associated with T2D [325].

Genetic manipulation of NOS isoforms in mice have allowed for interrogation of the mechanisms by which NO regulates metabolic health and disease. In mice, it has been reported that deletion of NOS3 causes insulin resistance, hyperlipidemia, and hypertension [326]. While full gene deletion mimics human “metabolic syndrome,” even partial gene deletion of NOS3 results in exaggerated insulin resistance, glucose intolerance, and hypertension induced by high fat feeding [327, 328]. A study using NOS3/NOS1 double-knockout mice showed that deletion of NOS3 promotes insulin resistance in both skeletal muscle and liver, whereas NOS1 deletion impairs insulin sensitivity only in liver [329]. Mice lacking all isoforms of NOS, i.e., NOS3/NOS1/NOS2 triple knockout mice, demonstrate increased visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance, and, it is interesting to note, that this is one of the few mouse strains to date to have spontaneous myocardial infarctions, apparently due to unstable coronary arteriosclerotic lesions [330]. That NOS is important to insulin sensitivity was further shown by studies in mice in which overexpression of dimethylarginine dimethylaminohydrolase—an enzyme that catalyzes the breakdown of the endogenous inhibitor of NOS, ADMA—increased insulin sensitivity [331].

Anti-obesogenic effects of NOS3

It appears that the metabolic phenotype elicited by insufficient levels of NOS3-derived NO relates directly to defects in intermediary metabolism in key peripheral tissues. Supporting evidence supplied by NOS3 KO mice include a markedly lower energy expenditure and decreases in mitochondrial content and fatty acid oxidation in muscle compared with WT mice [332]; and, as expected, NOS3 KO mice demonstrate an impaired ability to exercise [333]. Gain-of-function studies show a remarkable ability of NOS3 to regulate body composition and increase metabolism. Supplementation of NOS3 KO mice with nitrate, which can be serially reduced to nitrite and NO, reduced not only blood pressure, but visceral fat and triglycerides as well, thus reversing features of metabolic syndrome [334]. Furthermore, mice overexpressing NOS3 show an anti-obesogenic phenotype characterized by resistance to accumulation of white adipose tissue in response to a high fat diet, a higher metabolic rate, resistance to diet-induced hyperinsulinemia, and remarkably lower plasma levels of free fatty acids and triglycerides [335]. Similar results were obtained in an NOS3 phosphomimetic point mutant mouse model [336, 337]. Mutation of serine 1176 of NOS3 to an aspartic acid resulted in increased endothelial NO production as well as resistance to diet-induced weight gain and hyperinsulinemia; mutation of the residue to an alanine, which cannot be phosphorylated, resulted in insulin resistance and features of metabolic syndrome [157, 337].

How does NOS3 regulate metabolism and body composition? Several possibilities exist. Consistent changes in plasma lipids insinuate a central role of NOS3 in lipid oxidation or synthesis, e.g., NOS3 KO mice have elevated plasma levels triglycerides and free fatty acids compared with WT mice [326, 328], while NOS3 transgenic mice show diminished abundance of the lipids [122]. That these differences are due to modulation of fat oxidation capacity is suggested by studies showing a direct effect of NO on the capacity to oxidize fat. Not only do NOS3 KO mice show diminished fat oxidation capacity in skeletal muscle [332], but administration of a NOS inhibitor is sufficient to increase serum triglycerides and diminish hepatic fatty acid oxidation in rats [338], potentially by decreasing the activity of carnitine palmitoyl transferase [339]. Similar, NOS inhibitor-dependent decreases in fat oxidation capacity occur in heart [340]. In isolated hepatocytes, treatment with NO donors was shown to increase fatty acid oxidation in a cGMP-dependent manner by inhibiting acetyl CoA carboxylase (thereby decreasing production of malonyl CoA) and by stimulating carnitine palmitoyl transferase activity [341]. Interestingly, NO also inhibits fatty acid synthesis in hepatocytes [341], which is consistent with studies showing that NOS inhibitors [342] or genetic deletion of NOS3 increases lipid synthesis in liver [343]. In skeletal muscle, genetic deletion of NOS3 increases neolipogenic genes expression while downregulating genes involved in β-oxidation [332].

That genes involved in fatty acid oxidation are modulated by NO is consistent with data showing that overexpression of NOS3 increases expression of peroxisome proliferator activated receptor (PPAR)-α [122], which is well known to regulate lipid metabolism [344]. However, it is possible that NO regulates fat oxidation post-translationally as well. Recent studies show widespread S-nitrosation of multiple enzymes involved in intermediary metabolism. In particular, the liver enzyme, very long chain acyl-coA dehydrogenase (VLCAD) was shown to be nitrosated at Cys238, which increased the catalytic efficiency of the enzyme, and this modification was absent in NOS3 KO mice [345]. Collectively, these studies suggest that the powerful anti-obesity effects of NOS3-derived NO could be due to simultaneous increases and decreases in fat oxidation and synthesis, respectively.

NOS2 promotes insulin resistance

NO and RNS generated from the NOS2 enzyme could also regulate systemic metabolism. Most profound are its effects on insulin resistance. Although ablation of the NOS2 gene has no effect on weight gain, its absence was shown to improve glucose tolerance, normalize insulin sensitivity, and prevent derangements in PI3K/Akt signaling in high fat-fed mice [346]. Commonly, increases in NOS2 expression in skeletal muscle of obese mice are associated with increased S-nitrosation of the insulin receptor (IR), IR substrate-1 (IRS-1), and Akt, suggesting that nitrosative post-translational modifications of proteins in the insulin signaling pathway are responsible for NOS2-induced insulin resistance [347, 348]. The presence of NOS2 appears to decrease the abundance of IRS-1 via promoting its proteasomal degradation [148]. Interestingly, an acute bout of exercise was sufficient to downregulate NOS2 in high fat-fed rats as well as prevent S-nitrosation of proteins involved in insulin signaling, and administration of an inhibitor of NOS2 (L-N6-(1-iminoethyl)lysine; L-NIL) pheno-copied these effects [349]. Also, aspirin—which is one of the oldest known treatments for diabetes [350, 351] and improves blood glucose and insulin sensitivity in diabetic patients [352] and animal models of T2D [353]—inhibited NOS2-mediated S-nitrosation of IR, IRS-1, and Akt in skeletal muscle and improved insulin sensitivity [354].

In other tissues, NOS2 expression is also important for regulating insulin sensitivity. Selective overexpression of NOS2 in liver is sufficient to cause hepatic insulin resistance, hyperglycemia and hyperinsulinemia [355], and the use of an NOS2-specific inhibitor (L-NIL) reversed hyperglycemia, hyperinsulinemia, and insulin resistance in ob/ob mice [150]. In obesity, proinflammatory macrophages accumulating in adipose tissue are responsible for the majority of NOS2 expression [151-153] and may propagate the inflammatory signaling implicated in insulin resistance [130]. Importantly, the role of NOS2 in adipose tissue appears to differ remarkably from the canonical NO-cGMP pathway, as high fat diet-induced increases in proinflammatory cytokines and macrophage recruitment were attenuated by administration of sildenafil [356]. Interestingly, lack of NOS2 does not prevent age-induced insulin resistance [357], which suggests that not all insulin resistant states are created equal [358]. In line with this, mice lacking the NOS1 isoform show insulin resistance that appears to be due to a sympathetic, alpha-adrenergic mechanism [359].

6. Summary

Integration of results from these studies helps to form a model illustrating the role of NO in regulating obesity and insulin resistance (Figure 3). NO derived from NOS3 appears to have both anti-obesogenic and insulin-sensitizing properties. Its anti-obesogenic role stems from its ability to increase fat oxidation in peripheral tissues such as skeletal muscle, liver, and adipose tissue. As mentioned above, there is evidence that it also decreases lipid synthesis in liver. The impact of NOS3 on glucose metabolism and insulin sensitivity is underpinned by its capacity to increase transport of insulin and glucose to key peripheral tissues such as skeletal muscle and to regulate gluconeogenesis. Additionally, there may be implications for NOS3-mediated HISS release, which appears to enhance the vasodilatory properties of insulin. That NOS3 prevents hyperinsulinemia in two independent genetic gain-of-function studies [122, 337] further suggests that it could impact glucose metabolism by modulating insulin release. Other isoforms of NOS appear to promote deleterious changes in metabolism. In the brain, evidence suggests that NOS1-derived NO promotes hyperphagia. The NOS2 isoform promotes insulin resistance in both liver and skeletal muscle and is critical in inflammatory responses in multiple tissues, most notably, the adipose organ. Counter to NOS3, NOS2 appears to promote gluconeogenesis, and NOS2 has remarkable effects on cytokine-mediated insulin secretion. From these studies, it is apparent that NO is one of the most critical features regulating metabolism, body composition, and insulin sensitivity. Harnessing its beneficial metabolic actions is an exciting prospect for combatting metabolic disease.

Figure 3. Working model of the systemic effects of NO on obesity and metabolism.

Illustration of major organs and processes affected by NO and nitrogen oxides derived from NOS3, NOS1, and NOS2: The NOS3 isoform shows anti-obesogenic and insulin-sensitizing effects, which appears to be based in the ability of the enzyme to decrease lipid synthesis and promote fat oxidation in the liver and skeletal muscle. Additionally, NOS3 may be implicated in the secretion of hepatic insulin sensitizing substance (HISS), which might support insulin sensitivity in peripheral tissues such as skeletal muscle. NOS3 is important also for maximizing delivery of insulin and substrates to skeletal muscle, and this is likely critical in regulating insulin sensitivity and glucose tolerance. Through its actions in liver and pancreas, NOS3 may also suppress gluconeogenesis and prevent hyperinsulinemia, respectively. Additionally, NO increases the abundance of mitochondria and stimulates substrate oxidation capacity in adipose tissue, effectively promoting “browning” of white adipocytes. Conversely, other isoforms of NOS appear to have a more malevolent role in metabolism. NO derived from NOS1 promotes hyperphagia, and NOS2-derived nitrogen oxides can promote insulin resistance and inflammation in key peripheral tissues such as liver, skeletal muscle, and adipose tissue. In addition, NOS2 may affect glucose homeostasis by increasing glucose output from the liver and by impairing the endocrine activities of the pancreas.

HIGHLIGHTS.

• Obesity is a major pandemic of the 21^st century

• Decreased eNOS (NOS3) activity and abundance are commonly observed in obesity

• eNOS (NOS3)-derived NO has an anti-obesogenic effect

• iNOS (NOS2) promotes insulin resistance

• NO derived from iNOS (NOS1) appears to regulate appetite

NON-STANDARD ABBREVIATIONS AND ACRONYMS

3-NT

3-nitrotyrosine

ADMA

asymmetric dimethyl arginine

Akt

protein kinase B

AMP-activated protein kinase; BAT

brown adipose tissue

BH₂

dihydrobiopterin

BH₄

tetrahydrobiopterin

BMR

basal metabolic rate

Cav-1

caveolin-1

CCK

cholecystokinin

CDC

centers for disease control

cGMP

cyclic guanosine monophosphate

CoA

coenzyme A

EDRF

endothelial-derived relaxing factor

eEF1A1

elongation factor 1-α1

FAD

flavin adenine dinucleotide

FFA

free fatty acid

FMN

flavin mononucleotide

GSNO

nitrosated glutathione

HDL

high-density lipoprotein

HISS

hepatic insulin-sensitizing substance

HSP90

heat shock protein 90

IL-1β

interleukin-1 beta

IR

insulin receptor

IRS-1

insulin receptor substrate-1

Irs2

insulin receptor substrate 2

KO

knockout

L-NAME

L-NG-nitroarginine methyl ester

LNIL

L-N6-(1-iminoethyl)lysine

L-NMMA

NG-monomethyl-L-arginine

LDL

low-density lipoprotein

NADPH

nicotinamide adenine dinucleotide phosphate

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NMDA

N-methyl-D-aspartate

NO

nitric oxide

NOS1

neuronal nitric oxide synthase

NOS2

nitric oxide synthase 2

NOS3

nitric oxide synthase 3

NPY

neuropeptide Y

O-GlcNAc

O-linked N-acetylglucosamine

O₂^.−

superoxide

PGC-1α

PPAR-γ-coactivator-1α

PI3K

phosphoinositide 3-kinase

PKCβII

protein kinase C isoform βII

PPAR

peroxisome proliferator activated receptor

RNS

reactive nitrogen species

sGC

soluble guanylate cyclase

SIN-1

3-morpholinosydnonimine

SNO

S-nitrosothiol

STZ

Streptozotocin

T2D

type 2 diabetes

TLR

Toll-like receptor

TNFα

tumor necrosis factor α

UCP

uncoupling protein

VLCAD

very long chain acyl-coA dehydrogenase