Hypertension in obesity: is leptin the culprit?

Review Special issue: neural control of appetite Hypertension in obesity: is leptin the culprit? Stephanie E. Simonds and Michael A. Cowley Monash Obesity & Diabetes Institute, Department of Physiology, Monash University, Clayton, VIC, Australia The number of obese or overweight humans continues to increase worldwide. Hypertension is a serious disease that often develops in obesity, but it is not clear how obesity increases the risk of hypertension. However, both obesity and hypertension increase the risk of cardiovascular diseases (CVD). In this review, we examine how obesity may increase the risk of developing hypertension. Specifically, we discuss how the adipose-derived hormone leptin influences the sympathetic nervous system (SNS), through actions in the brain to elevate energy expenditure (EE) while also contributing to hypertension in obesity. Introduction Energy homeostasis is a tightly regulated balance of caloric intake and EE. Humans become overweight or obese when intake exceeds expenditure. Although no single cause has been identified as the factor resulting in a mismatch of intake and expenditure in modern societies, it is clear that the prevalence of obesity continues to increase in both the developed and the developing world [1]. Although obesity is linked to increased mortality, the accumulation of excess body fat is not the direct cause of death. Rather, obesity increases the risk of a variety of diseases that can cause death and disability. Serious diseases comorbid with obesity include type 2 diabetes mellitus, cancers, sleep apnea, musculoskeletal disorders, hypertension and CVDs. CVD is the number one cause of death globally; it also causes significant morbidity and imposes high healthcare costs [2 4]. The presence of hypertension increases the risk for the development of CVDs, including heart disease and renal failure [5]. Obesity and hypertension together further increase the risk of mortality from CVD [6]. Leptin is an adipose-derived hormone that reduces body weight and is substantially elevated in obesity [7]. However, leptin resistance develops in obesity and restricts the ability of leptin to reduce weight, although it retains the capacity to increase sympathetic nerve activity (SNA); we propose here that this increased SNA increases blood pressure (BP) in obesity [8 11]. Hypertension Hypertension is defined as chronically elevated BP in the arteries and is diagnosed when systolic BP (SBP) is constantly registered above 140 mmhg and diastolic BP Corresponding author: Cowley, M.A. (Michael.Cowley@monash.edu). Keywords: hypertension; obesity; leptin; sympathetic nerve activity; hypothalamus; dorsomedial hypothalamus. (DBP) above 90 mmhg [12 14]. Hypertension causes chronically increased pressure on multiple organs, and is a major cause of end-organ damage [15]. Hypertension strongly predicts an increased risk of CVDs. Although hypertension is associated with obesity, we do not yet understand mechanistically how obesity causes a chronic elevation in BP. Chronically elevated BP develops when the kidneys fail to regulate renal pressure natriuresis correctly, resulting in greater sodium reabsorption and, hence, fluid retention [16]. Several factors are potentially involved in the development of hypertension, including renal damage, function of the autonomic nervous system (ANS), including increased SNA, activation of the renin angiotensin system, and the physical compression of the kidneys [17]. Renal compression Renal compression is the development of additional pressure on the kidneys or renal artery. Compression of a kidney can cause hypertension, as demonstrated by wrapping dog kidneys in cellophane [18]. After the kidneys were bilaterally wrapped in cellophane, BP increased within 2 weeks post surgery and continued to increase for several months until it peaked (as high as 240 mmhg) and Glossary Leptin resistance: leptin levels are generally elevated in obesity, yet leptin fails to promote actions to reduce body weight. Research suggests the ARH neurons in obese mice fail to respond to leptin and promote neuropeptide changes that decrease food intake, increase EE, and reduce body weight [8,97]. Obese humans are leptin resistant, as seen by the failure of recombinant leptin to cause weight loss, but the mechanisms of this leptin resistance in humans are less well understood [8]. Despite this inability of ARH neurons to respond to leptin in obesity, leptin can still increase SNA through actions in the brain. Thus, it is hypothesized, supported by immunohistochemistry staining using a marker of lepr activation (pstat3), that leptin resistance is selective to the ARH, with other hypothalamic and brain regions remaining responsive to leptin in obesity [68,79]. Pressure natriuresis: the increase in sodium excretion and, therefore, water excretion by the kidneys as urine, when BP increases. Loss of fluid as urine decreases fluid volume and, therefore, BP. Increased SNA can shift pressure natriuresis to higher pressures. A shift in pressure natriuresis as occurs in hypertension means that the kidney will balance fluid resorption around a higher BP, therefore maintaining hypertension. Sympathectomy: the removal of the sympathetic ganglia was more commonly used to treat chronically elevated BP before effective drugs were available. This treatment was effective in lowering BP, although BP did revert to the elevated level over time [45]. As with all surgery, sympathectomy is a risky procedure and its use as a first-line treatment against elevated BP was reduced after safe, effective, antihypertensive drugs became widely available. However, currently available drugs are not an effective treatment for all forms of hypertension and a new procedure to remove sympathetic innervation of the kidney via the renal artery has been developed. 0166-2236/$ see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tins.2013.01.004 Trends in Neurosciences, February 2013, Vol. 36, No. 2 121

remained elevated [18]. When only one kidney was compressed, the resultant hypertension was not as severe. Removal of the nerves innervating the kidney did not prevent hypertension developing, demonstrating that hypertension induced by renal compression is independent of the SNS [18]. Subsequent studies confirmed that renal compression can contribute to hypertension in a variety of species, including dogs, rabbits, and rodents [19]. Human case studies have reported the development of hypertension as a consequence of renal compression secondary to other diseases, suggesting that renal compression is one mechanism by which hypertension develops [20,21]. Renal damage Damage to renal function, in particular renal fibrosis, can develop and contribute to the development of hypertension. Kidneys from hypertensive dogs transplanted into normotensive dogs resulted in the recipient dogs subsequently developing hypertension; however, hypertension did not develop if the donor kidney did not originate from a hypertensive dog [22 24]. Further studies established that this hypertension develops in association with glomerular hyperfiltration [25,26]. The role of the ANS in hypertension The ANS consists of two arms (excluding the enteric nervous system, which exclusively controls the gastrointestinal systems): the parasympathetic nervous system (PSNS) and SNS. The PSNS is predominantly involved in actions while the body is at rest, including digestion, urination, and sexual arousal. The SNS is involved in the regulation of body homeostasis, actions including the fight or flight responses, and vasoconstriction. Autonomic tone is usually balanced between the two systems; however, each arm of the ANS does not equally stimulate or inhibit every organ to the same extent. For example, SNA is not globally increased to every target in response to a stimulus (i.e., a hormone). Some stimuli increase SNA to the kidney while leaving the SNA to the heart unchanged; similar phenomena exist in the PSNS. The ANS plays a significant role in balancing energy homeostasis as well as regulating cardiovascular functions. During periods of weight loss, the ANS acts to prevent further weight loss and, hence, the PSNS increases activity and the SNS decreases activity [27]. Reduced SNS tone leads to reduced stimulation of brown fat, and reduced futile cycling in fat, leading to less heat production and less energy consumption. This diminishes weight loss and may also account for some of the beneficial reduction in BP and cardiovascular risk associated with weight loss. Weight gain causes the ANS to increase EE to try and drive weight loss, the PSNS decreases in activity, and the SNS increases, even in obesity [27]. The role of the PSNS in hypertension The PSNS, similar to the SNS, propagates messages from the brain to the periphery. Both the pre- and postganglionic parasympathetic nerves are cholinergic and stem from cranial nerves or closer to the S2 S4 region of the spine (Figure 1). Cranial nerve 10 (X) (also known as the vagus nerve) controls parasympathetic innervation of key peripheral organs, including the heart, stomach, pancreas, liver, and lungs. The brain controls the activity of the PSNS, with the nucleus ambiguous (NA) and dorsal motor nucleus of the vagus nerve (DMX) regions known to send projections to preganglionic neurons (Figure 2). Numerous inputs from many brain nuclei send projections or relayed projections to these two nuclei and, hence, impact the activity of the PSNS (Figure 2). The PSNS has the capacity to counteract some of the actions of the SNS, including reducing heart rate (HR) and contractility. This causes reduced cardiac output and reduces the circulation of blood throughout the body, independent of changes to blood volumes. In hypertension, it is hypothesized that the activity of the PSNS decreases and, hence, enables the SNS to further increase BP and HR. It is known that patients with borderline hypertension have increased sympathetic and decreased parasympathetic tone [28]. It is challenging to measure parasympathetic tone reliably and this may have hampered research into the parasympathetic basis of hypertension. The role of the SNS in hypertension The SNS contributes to the homeostatic balance of the body [29]. The central nervous system (CNS) provides inputs into the SNS, which begins with nerves extending from the intermediolateral cell column [intermediolateral nucleus (IML)] of the spinal cord. It is a region of gray mater extending from thoracic region T1, to lumbar region L3 that contains autonomic motor neurons, the sympathetic preganglionic nerves. Both sympathetic and parasympathetic nerve fibers are cholinergic, whereas the sympathetic post ganglionic nerve fibers are adrenergic. From the IML, the SNS preganglionic nerves run a short distance from the spinal cord to the sympathetic trunk, where they synapse with sympathetic postganglionic nerves that innervate many organs of the body (Figure 1). The adrenal gland receives direct preganglionic sympathetic innervation. The SNS is controlled centrally from inputs from premotor neurons in numerous brain regions [including raphe pallidus (RPa), locus coeruleus (LC), paraventricular nucleus of the hypothalamus (PVH), ventrolateral A5 region of the pons (A5), and the rostral ventrolateral medulla (RVLM)], which receive direct inputs from an array of other nuclei around the brain, including regions in the hypothalamus and the nucleus of the solitary tract (NTS) (Figure 3) [30,31]. Inputs from the periphery and other brain regions are known to have an influence on the activity of the SNS. The SNS is essential in mediating EE, including thermogenesis, in the control of energy balance. This is eloquently demonstrated in research showing that b adrenergic-knockout mice are unable to regulate bodyweight and thermogenesis, due to an inability of the SNS to covey its message to target organs [32]. In addition, mice lacking the ability to produce catecholamines (SNA products) also lack the ability to increase thermogenesis in response to cold conditions [33]. Thermogenesis can occur in muscle and in brown adipose tissue (BAT) deposits. In BAT, specialized adipose cells containing a high density of mitochondria are used for heat production, rather than for energy storage. BAT is exclusively innervated by the SNS and, hence, the regulation of thermogenesis is dependent on the SNS [34 36]. Increased SNA in obesity or in response to cold causes an 122

Review Trends in Neurosciences February 2013, Vol. 36, No. 2 Eye Lacrimal gland Nasal mucosa Paro d gland Parasympathe c Sympathe c 1 2 3 Salivary gland Trachea 4 III VII IX Cranial nerves Lung T1 T2 T3 T4 Thoracic BAT X T5 Heart Spinal nerves T6 Liver T7 Stomach Spleen T8 5 T9 T10 6 T11 S2 T12 Gall Bladder Splanchnic nerves Pancreas S3 Adrenal gland S4 L1 L2 L3 7 Spinal nerves 8 Kidney Colon Sympathe c trunk Small intes ne Key: Rectum WAT Urinary bladder Parasympathe c preganglionic fibre Parasympathe c postganglionic fibre Sympathe c preganglionic fibre Genitalia Sympathe c postganglionic fibre TRENDS in Neurosciences Figure 1. The autonomic nervous system: pathways from the brain to the periphery of the sympathetic and parasympathetic nervous systems. The parasympathetic nerves leave the brain and preganglionic cholinergic parasympathetic nerves (red) (the third, seventh, ninth and tenth cranial nerves, and spinal nerves S2, S3, and S4) innervate target organs. The third (III) parasympathetic preganglionic nerve synapses to the postganglionic parasympathetic nerve in the ciliary (1). The seventh (VII) parasympathetic preganglionic nerve synapses to the postganglionic sympathetic parasympathetic nerve in either the pterygopalatine ganglion (2) or the submandibular region (3). The ninth cranial nerve (IX) parasympathetic preganglionic nerve synapses in the optic ganglia (4). The parasympathetic preganglionic nerve axons of the vagus nerve are long and this leads to the synapses of terminals onto parasympathetic postganglionic synapses (purple) close to target organs. The short parasympathetic postganglionic nerves are also cholinergic and they release acetylcholine at their terminals, propagating the parasympathetic nerve message from the brain. The vagus nerve (cranial nerve X) is a well-studied parasympathetic nerve because it innervates numerous abdominal organs, including esophagus, trachea, heart, lungs, stomach, pancreas, liver, and kidney. It also appears to regulate the activity of white adipose tissue (WAT), although controversy exists about the impact of parasympathetic innervation of WAT [133 135]. The sympathetic nerves arise from the intermediolateral nucleus (IML) of the spine from T1 to L3; the preganglionic sympathetic nerves (blue) are cholinergic. Sympathetic preganglionic nerves are shorter than parasympathetic preganglionic nerves and synapse onto adrenergic sympathetic postganglionic nerves (green) in the sympathetic trunk, a set of ganglia that sit adjacent to the spine. These postganglionic nerves release catecholamine products that act on these target organs. Interestingly, two organs that receive sympathetic inputs, the adrenal and sweat glands, are innervated with acetylcholine acting at muscarinic receptors, rather than with catecholamine as the product. The T5 T9 region synapses sympathetic preganglionic nerves to sympathetic postganglionic nerves in the suprarenal ganglia (celiac ganglia) (5). The T10 T12 sympathetic preganglionic nerves synapse to sympathetic postganglionic nerves in either the aorticorenal (6) or superior mesenteric ganglia (7). The L1 L2 region sympathetic postganglionic nerves synapse to postganglionic sympathetic nerves in the inferior mesenteric ganglia (8). These sympathetic postganglionic nerves innervate many organs in the body, including the heart, kidney, blood vessels, lungs, liver, and pancreas. Sympathetic nerves also innervate (originating from along the sympathetic trunk, depending on the location of the fat deposit in the body) and regulate WAT. Only the sympathetic nervous system regulates the activity of brown adipose tissue (BAT), which is located in multiple small clusters, including thoracic, paraspinal, subclavicular and interscapular regions. Many organs receive both sympathetic and parasympathetic inputs, showing the ability of these systems to regulate homeostasis cooperatively within the body. 123

Prl abst PBN VMH PVH DMH LH ARH NTS DMX NA Key: Lep n receptor Insulin receptor Melanocor n 4 receptor (MC4R) Parasympathe c nerve ouflow cranial nerve (vagus nerve X) to target organs TRENDS in Neurosciences Figure 2. Brain circuitry of the parasympathetic nervous system (PSNS). Schematic of major brain nuclei that regulate PSNS efferent tone. The NA and DMX (purple nuclei) are both key regions involved in the activation of the PSNS, especially the vagus nerve (green lines). Several other regions, including the NTS, PVH, abst, and Prl, send direct inputs (blue lines) to the DMX and NA. The hypothalamus (i.e., The DMH, ARH, VMH, and LH) provides inputs directly into the PVH and NTS and, hence, has the potential to influence parasympathetic output. Leptin receptors (yellow circles), insulin receptors (orange circles), and melanocortin 4 receptors (green circles) are expressed in several of these nuclei. Abbreviations: abst, anterior bed nucleus of the stria terminalis; ARH, arcuate nucleus of the hypothalamus; DMH, dorsomedial hypothalamus; DMX, dorsal motor nucleus of the hypothalamus; NA, nucleus ambiguous; NTS, nucleus of the solitary tract: PVH, paraventricular hypothalamus: Prl, prelimbic cortex. increased release of noradrenaline, and agonism of b3 adrenergic receptors (b1 adrenergic receptors may also play a role) [37 39]. This activates the G protein-coupled adenylyl cyclase/camp/protein kinase, leading to the lipolysis of triglycerides to generate free fatty acids (FFA) [38,39]. Increased FFA activates uncoupling protein 1 (UCP1) and therefore increases the production of heat. UCP1 is critical for the production of heat by BAT cells, because UCP1 dissipates the proton gradient that is produced during oxidative phosphorylation, forming heat rather than ATP [38,40]. The other mechanism to expend energy via thermogenesis is via muscle. Activation of SN or agonists that mimic SNA, including isoprenaline, increase muscle temperature, although the mechanisms of this effect are not fully understood [41]. Sympathetic nerves innervate the heart to control cardiac pace and contractility. Increased SNA can override pacemaker cells and cause the heart to beat faster and with a greater force (contractility). The SNS innervates blood vessels and causes constriction, reducing the luminal diameter and therefore increasing BP. The SNS also innervates the kidneys, which are critical in the control of fluid balance within the body and, hence, in the regulation of BP. Sympathetic innervation of the kidney can change multiple functions and, when all combined, can elevate BP by shifting the pressure natriuresis curve to higher pressures. A key finding illustrating the involvement of the SNS in the control of hypertension is that removal of the sympathetic nerves to the kidney (denervation) reduced BP in hypertensive animals [42,43]. In dogs fed a short-term 124

IL abst PBN CeA VMH PVH ARH DMH LH A5 LC RPa NTS RVLM CVLM IML Key: Lep n receptor Insulin receptor Melanocor n 4 receptor (MC4R) Sympathe c nerve ou low to target organs TRENDS in Neurosciences Figure 3. Brain circuitry of the sympathetic nervous system. Schematic of major brain nuclei that regulate sympathetic nervous system tone. The IML receives direct inputs from numerous nuclei that contain premotor sympathetic cells (red lines). These regions include the RPa, PVH, RVLM, A5, and LC (dark-pink circles). Other brain regions influence the activity of these regions by sending excitatory and inhibitory inputs. Several of these regions are from the hypothalamus, including the DMH, ARH, VMH, and LH. Extra hypothalamic regions also play critical roles, including the CeA, IL, PBN, and NTS. Leptin receptors (yellow circles), insulin receptors (orange circles), and melanocortin 4 receptors (green circles) are expressed in several of these nuclei. Abbreviations: abst, anterior bed nucleus of the stria terminalis; ARH, arcuate nucleus of the hypothalamus; A5, A5 adrenergic neurons; CeA, central amygdale; CVLM, caudal ventrolateral medulla; DMH, dorsomedial hypothalamus; IL, infralimbic cortex, IML, intermediolateral nucleus; LC, locus coeruleus; LH, lateral hypothalamus; NA, nucleus ambiguous; NTS, nucleus of the solitary tract; PBN, parabranchial nucleus; PVH, paraventricular hypothalamus; RPa, ralph pallidus; RVLM, rostral ventrolateral medulla; VMH, ventromedial hypothalamus. high-fat diet (HFD), despite similar weight gain, BP only increased in the dogs with intact renal nerves [44]. Previously, when limited medication for effective treatment of BP was available, surgical intervention (i.e., sympathectomy) was used more readily [45]. Recently, a modified sympathectomy procedure has gained acceptance, and is becoming more widely used to treat refractory hypertension [46]. Ardian Inc. (Palo Alto, California) has developed a catheter-based procedure where the renal nerves can be destroyed by radio frequency ablation through the renal arterial wall and, hence, the sympathetic nerve input into the kidney can be disrupted [46,47]. The therapy appears to be effective in reducing BP in humans, and the decrease in BP has been sustained in patients for at least 1 year. Research is now currently being conducted into treating other forms of hypertension, including hypertension that develops in obesity, with this catheter-based approach. Although this treatment is a quick key hole procedure, it would be unfeasible to treat all patients with hypertension via a surgical intervention, just as it is unfeasible to treat all patients with obesity via surgical intervention (i.e., Roux en Y gastric bypass, lap band, or gastrectomy surgeries). Elevated SNA correlates with total body noradrenaline concentration, and is elevated in normotensive subjects who are members of a family with a history of hypertension [48,49]. Therefore, at both a scientific level and treatment development perspective, understanding how hypertension specifically develops in obesity could deliver more effective treatment in the future. Why is hypertension more likely to develop in individuals who are overweight? Animal models demonstrate that both compression of the kidney and altered ANS function could play key roles in the development of hypertension. 125

Could hypertension develop in obesity as a consequence of renal compression? In obesity, the kidney can become compressed because it can become completely surrounded by adipose tissue [50]. Although the capsule surrounding the kidney is generally of low pressure, additional pressure from surrounding fat can increase the intrarenal pressure. Obese dogs have increased renal interstitial fluid pressure compared with lean dogs [51]. Obese individuals have increased abdominal pressure compared with patients who are nonobese [52]. The sagittal diameter of the abdominal region correlates with intra-abdominal pressures [52]. Increased abdominal fat accumulation and circumference are known to exacerbate the risk for the development of hypertension and CVDs, therefore compression of the kidney and increases in intra-abdominal pressure might be key to worsening hypertension in obesity [53,54]. Although compression of the kidney can elevate BP, the process is slow, especially if it due to the accumulation of fat surrounding the kidney; however, this does not explain how weight loss can cause immediate reductions on BP, especially because perirenal fat does not decrease quickly with weight loss. Therefore, it is unlikely that compression of the kidney is the primary cause of hypertension in obesity. Another fact that discounts renal compression as a primary cause in the development of hypertension in obesity, is that severely obese mice (eg. ob/ob mice), have compressed kidneys but do not develop hypertension, indicating the involvement of other factors [55,56]. Could hypertension develop in obesity as a consequence of dysregulation of the PSNS? In obesity, the SNS appears to be a key factor in the development of many aspects of the metabolic syndrome. There may also be a role for the PSNS. However, in obesity, little research points to the PSNS being heavily involved in chronically elevated BP. PSNA, at least to the heart, is decreased in obesity [27,57]. The DMX region is critical in propagating the activity of the PSNS, and the melanocortin 4 receptor (MC4R) is heavily expressed in the DMX and adjacent NTS [58 60]. It is also known that agonists of the MC4R can acutely increase BP in rodents and humans [61 63]. However, a recent study showed that re-expression of MC4R on only preganglionic parasympathetic cholinergic neurons had little impact on EE, contrary to what is seen with preganglionic sympathetic cholinergic neurons [64]. Although this research does not exclude a role for the PSNS in controlling BP, it highlights the need for more research to determine the true effects of obesity on the PSNS and also its involvement in cardiovascular regulation and EE. Does the SNS cause hypertension in obesity? For many years, it was thought that obesity developed due to an inability of the body to increase EE and, therefore, that obesity developed as a consequence of reduced SNA [65,66]. However, over the past few decades, this hypothesis has been refuted. Research now shows that the SNS is generally overactive in obesity, which is thought to be an attempt to elevate EE to cause weight loss [67]. Obese animals have increased BAT temperature compared with control animals and a b3 adrenergic blocker can reduce this increase in BAT temperature, demonstrating that the increased thermogenesis occurring in obesity is mediated by the SNS [68]. SNA (as measured by noradrenaline spillover), including that to the muscles, is elevated in obese humans, and weight loss reduces these parameters [10,65,69 71]. Studies in dogs support this research, with renal denervation protecting dogs from developing elevated BP when fed a HFD [44]. The role of leptin in the regulation of the SNS First-line treatment for both obesity and hypertension is weight loss, but is there a common molecular link between these two conditions? Such a link may involve the adiposederived hormone leptin [72] (Table 1). Leptin is an adipokine that has the capacity to both decrease food intake and elevate SNA [9,73,74]. The concentration of leptin in the plasma is proportional to the mass of adipose tissue and leptin can increase locomotor activity, EE, and thermogenesis [7,68,73,75,76]. Leptin-deficient ob/ob mice exhibit reduced locomotor activity at baseline, but exogenous administration of leptin can increase their locomotor activity over a sustained period [75]. Leptin can also increase thermogenesis due to its ability to increase SNA to BAT deposits [9]. Leptin-deficient ob/ob mice have reduced BAT temperature compared with wild type mice and diet-induced obese mice [68]. Administration of leptin to ob/ob mice elevated BAT temperature (also indirectly measured via UCP1 expression) and core body temperature [68,76,77]. Obese leptin-deficient humans also exhibit reduced core body temperature, most likely due to decreased SNA, as demonstrated when challenged by cold conditions [78]. Although how leptin enters the brain and finds its way to receptors is still debated, it is widely accepted based on immunohistochemistry results that leptin acts in the brain, sometimes in sites far from circumventricular zones, stimulating activity of signaling pathways, including phosphorylated signal transducer and activator of transcription 3 (pstat3), a common marker of leptin receptor (lepr) activation [79]. Through actions in the brain [specifically the dorsomedial nucleus of the hypothalamus (DMH)], leptin has been demonstrated to increase SNA to BAT cells, increasing BAT temperature and UCP1 expression [68,80,81]. One interesting finding is that SNA in obesity is not restricted to BAT deposits and is increased to not only other thermoregulating organs including muscle, but also to cardiovascular regulatory tissues, such as the kidney and heart [10,11]. Leptin administered to rodents can elevate SNA and increase HR and BP in a dose-dependent manner [9,74]. Mice with elevated plasma leptin levels have increased BP [9,11]. Plasma leptin concentration positively correlates with the renal SNA (rsna) in humans and also with the development of hypertension in humans [82,83]. Recent studies have shown that leptin produces many of its effects through initial actions at neurons in the hypothalamus. Surprisingly, leprs are not located in the one hypothalamic brain region, the PVH, that contains premotor neurons (i.e., neurons with the ability to increase SNA directly); thus, the ability of leptin to increase SNA must first be relayed via other regions [84]. LepR-expressing 126

Table 1. Timeline of key developments related to the involvement of leptin in increasing SNA and contributing to the development of hypertension in obesity Year Major finding Species Implication Refs 1994 Identification of the ob gene product, leptin Mouse and human 1995 1999 Exogenous administration of leptin can decrease food intake and subsequently body weight Plasma leptin levels positively correlate with body fat and BMI Leptin administration increases locomotor activity, metabolic rate, and core body temperature Identification of a key signaling molecule associated with obesity Mouse Leptin is produced in mice to regulate body weight. Mice that lack leptin (ob/ob) develop obesity. This obesity can be cured by leptin administration Mouse and Human obesity is not a consequence of leptin humans deficiency Mouse Key role for leptin in body weight regulation through physiological changes additional to decreased food intake Identification of the db gene, receptor for leptin Mouse Identification of a key signaling receptor critical for mediating the actions of leptin Leptin increases noradrenaline depletion from fat deposits Peripheral leptin administration increased SNA to BAT, kidney, adrenal gland, and the hindlimb in lean rats Renal noradrenaline spillover positively correlates with BMI Chronic leptin infusion into obese animals increased HR and BP in lean rats Mice lacking the ability to produce noradrenaline were unable to mediate leptin-induced increases in UCP1, (marker of BAT activation) The melanocortin system can increase SNA; leptin-mediated increases in renal SNA occur at least partially through the melanocortin system in lean rats Leptin causes limited weight loss in obese patients Leptin-deficient ob/ob mice, despite obesity, do not exhibit elevated BP 2000 2004 Transgenic skinny mice that overexpress leptin exhibit elevated BP Renal noradrenaline spillover positively correlates with plasma leptin concentration Leptin acting on neurons in the VMH and DMH increases cardiovascular parameters in lean rats The melanocortin system mediates cardiovascular, renal, and metabolic actions of leptin in lean rats Leptin resistance in obesity is restricted to lepr-expressing neurons in the ARH 2005 2009 Leptin is unable to cause weight loss in obesity; however, it can still increase SNA and elevate BP MC4R knockout mice are not hypertensive despite exhibiting obesity and hyperleptinemia Leptin resistance develops in neurons of the ARH, causing an inability of leptin to cause the secretion of neuropeptides, hence the melanocortin pathway is not active in obesity Humans with polymorphisms of the lepr exhibit reduced SNA The importance of the melanocortin system in the progression of elevated blood pressure in obesity Mouse Leptin may cause weight loss through decreased food intake and additionally through increased SNA Rats Leptin acting via the lepr has the capacity to increase SNA to numerous organs around the body Humans In obesity, noradrenaline spillover significantly elevated, hence SNA elevated in obesity Rats Leptin increases BP, most likely through activation of the SNS Mouse Leptin requires noradrenaline to mediate its thermogenetic effects, indicating activation of the SNS Rats Identification of the systems leptin uses to increase SNA Humans Suggestion of leptin resistance in obese humans. Leptin unlikely to be an anti-obesity treatment Mouse Leptin levels and not excess body weight correlate with hypertension in obesity Mouse Body fat levels do not have to be elevated to cause hypertension, elevated plasma leptin levels can cause it Humans Leptin could be the cause of increased noradrenaline spillover, hence SNA in obesity is likely to cause development of hypertension Rats Key hypothalamic regions, including the VMH and DMH, are involved in the ability of leptin to alter cardiovascular parameters Rats Identification of the systems leptin uses to increase sympathetic nerve activity and increase BP Mouse Demonstrated regional specificity of leptin resistance Mouse Identification that leptin retains the ability to induce hypertension in obesity despite leptin resistance Mouse Despite obesity, an intact melanocortin system is required for hypertension to develop in obesity Mouse Identification of an inactive melanocortin system in obesity Human Intact leptin circuitry is required for increased SNA. Important role of leptin, as shown in rodent studies, translates to humans Human Mutations in the melanocortin system lessen the risk of hypertension developing in obesity [72] [73] [7] [130] [131] [132] [9] [10] [74] [33] [62] [8] [55] [100] [82] [108] [104] [79] [11] [63] [97] [101] [61] 127

Table 1 (Continued ) Year Major finding Species Implication Refs 2010 Removal of leprs from POMC neurons reduces a leptin-mediated increase in BP Mouse Leptin can act through POMC to induce elevated BP, at least in lean mice [106] Leptin-mediated activation of BAT is through neurons in the DMH. An independent pathway to the melanocortin system is also involved Removal of lepr from the ARH removes the leptin-mediated increase in SNA LepR-expressing neurons in the DMH are critical in the regulation of BAT activity MC4R agonists produce physiological results that, unlike previously developed agonists, cause weight loss without elevating BP Mouse Leptin has the capacity to increase SNA and thermogenesis in obesity, independent of the melanocortin system Mouse Leptin acts through neurons in the ARH in obesity to increase SNA Mouse Identification of the DMH region in leptinmediated actions, including SNA-mediated thermogenesis in BAT Rhesus macaques Identification of the MC4R agonist that can cause weight loss without increasing cardiovascular parameters; gives hope to this circuitry being manipulated in obesity treatment [68] [107] [114] [90] neurons are located in multiple nuclei throughout the hypothalamus, including the arcuate nucleus of the hypothalamus (ARH) [84]. The ARH contains two distinct neuronal populations that express leprs [proopiomelanocortin (POMC) and Agouti-related peptide/neuropeptide Y (AgRP/NPY)]. A third population, rat insulin-2 promotor (RIP) cells, has recently been the focus of interesting research. However, because their neuropeptide characteristics are unknown, and the promoter, insulin, is questionably made in the brain, the exact physiological role of these neurons in the ARH is uncertain [85]. The food intake and EE actions of leptin are substantially mediated via ARH neurons. POMC neurons are depolarized by leptin and their activation causes decreased food intake and increased EE [86,87]. Recently, lepr on POMC neurons have been demonstrated to be critical in regulating glucose homeostasis, but to be less important in mediating energy homeostasis [87]. Alpha melanocyte-stimulating hormone (a-msh) is a key neuropeptide released from POMC neurons that agonizes MC4R and MC3R in the brain, leading to decreased food intake and increased SNA and EE [88,89]. The melanocortin system has been a major target of obesity treatments because its dysfunction is implicated in human obesity; thus, melanocortin-specific agonists for the MC4R have been developed to treat obesity. These agonists reduce body weight in many species [61,88,90]. However, most agonists of the MC4R also increase SNA to the kidney and heart. Thus, although such agonists decrease body weight, they also exacerbate hypertension. The other neuronal population that express leprs in the ARH are AgRP/NPY neurons, which are hyperpolarized by leptin [91]. AgRP antagonizes the MC4R and, hence, opposes a-msh actions [88]. Inhibition of NPY/AgRP neurons also decreases a tonic GABAeric input into POMC neurons, further increasing the release of POMC products upon POMC activation [86,92]. NPY binds to Y1 and Y5 receptors in the brain independent of the melanocortin system. It increases food intake and has the capacity to decrease SNA, probably to decrease EE [93 96]. Leptin resistance develops in obesity In lean animals, neurons in the ARH detect leptin and function reciprocally to regulate energy homeostasis. By contrast, in obese mice, these neurons fail to respond to leptin and, hence, do not regulate energy homeostasis [68,79,97,98]. Interestingly, despite this leptin resistance and an inability of leptin to decrease food intake, it retains the ability in obese animals to increase SNA to numerous tissues, including BAT deposits, lumbar nerve, muscle, and the kidneys [11,68,71,99]. This increase in SNA is probably an attempt by the body to expend energy and rebalance body weight, although this increase in EE is not sufficient to oppose the increase in calorie intake. Despite failing to regulate food intake and body weight, leptin appears to have a significant role in the development of high BP in obesity [99]. Transgenic mice that overexpress leptin are lean yet have elevated BP compared with weight-matched controls [100]. Diet-induced obese mice are hyperleptinemic and have elevated SNA and BP [11,99]. Human studies show a positive correlation between plasma leptin concentration and BP [83]. Polymorphisms in the lepr have also been associated with increased plasma leptin concentration, increased body mass index (BMI), and decreased whole-body noradrenaline spillover [101]. In obesity, leptin has substantial actions independent of the melanocortin system Diet-induced obese mice, with elevated plasma leptin concentration due to increased fat deposits, have chronically increased BAT temperature and UCP1 expression [68]. This is despite leptin resistance in the ARH [68,79,97]. Leptin fails to increase the release of a-msh and to inhibit secretion of AgRP and NPY in the hypothalamus of obese mice [97]. Activation of the melanocortin system can mediate an increase in BAT temperature in lean animals, but mice that lack MC4R expression also have elevated BAT temperature. Hence, it appears that, in obesity, a melanocortin-independent pathway may link leptin to control of BAT temperature [68]. Leptin actions via the melanocortin pathway appear to regulate BP and HR in lean animals, but whether the melanocortin pathway regulates leptin effects in obesity remains debatable. Agonists of the melanocortin system suppress food intake and increase both BP and HR in lean and HFD-fed rodents [89,97,102]. Antagonism of melanocortin receptors increases food intake and causes substantial weight gain in lean and overweight rats, and decreases 128

BP to a greater extent in overweight rats with elevated BP [103,104]. Mice and humans with MC4R deficiency exhibit elevated plasma leptin concentrations, have reduced HR, and a reduced incidence of hypertension, despite moderate obesity [61,63]. Humans that are heterozygous carriers of MC4R mutations exhibit lower muscle SNA and demonstrate an inverse correlation between their muscle SNA and BMI, as well as between muscle SNA and plasma leptin concentration [105]. Removal of leprs from POMC neurons in lean mice attenuated the ability of exogenous leptin to increase BP and decrease plasma glucose and insulin levels [87,106]. A further study removed leprs from the entire ARH of moderately overweight mice (30 g compared with controls weighing approximately 26 g) that were feed a HFD. Such manipulation attenuated leptinmediated increases in renal and BAT SNA and BP [107]. Questions still remain regarding the involvement of ARH neurons in obesity and, therefore, the involvement of the melanocortin system in leptin-mediated hypertension and EE. There is good evidence that the melanocortin pathways mediate the effects of leptin in lean animals [104,106]. However, this has not been thoroughly tested in very obese mice that are leptin resistant with inactive arcuate melanocortin pathway signaling [68,79,97]. Are other hypothalamic regions involved in leptin-mediated hypertension? Weight gain reduces the ability of leptin to regulate neuronal activity in the ARH [97]. Despite the melanocortin pathway being implicated in the development of hypertension in obesity, hypertension in response to leptin may develop due to an entirely different brain region and different neuronal circuitry. LepR-expressing neurons in other hypothalamic nuclei, including those neighboring the ARH, remain responsive to leptin in obesity [68,79]. The ventral medial nucleus of the hypothalamus (VMH) and DMH each contain large concentrations of lepr-expressing cells [84]. Microinjection of leptin into the VMH has been demonstrated to elevate BP and rsna, whereas microinjection into the DMH increased both BP and HR [84,108]. Interestingly, the PVN contains very few leprs [84]. Microinjection of leptin into the PVN produces no change in cardiovascular parameters, highlighting the DMH and VMH as possible areas of interest in leptin-mediated increases in SNA and cardiovascular parameters [79,108]. This is especially true in obesity, because these regions remain leptin responsive. It has been known for many years that the DMH is involved in mediating sympathetic outflow, especially to BAT [80,109]. LepR-expressing neurons in the DMH project directly to the PVN, and neurons in the DMH also project to the RVLM and RP. Such regions all contain sympathetic premotor neurons with the capacity to increase SNA directly to target organs (Figure 3) [39,58, 110 113]. It has recently been demonstrated that coldinduced thermogenesis causes neuronal activation in a population of lepr-expressing DMH neurons [114]. These neurons then send projections to the RP and contribute to increase thermogenesis [114]. Leptin administration into the DMH can mediate thermogenesis in both lean and dietinduced obese mice and can increase UCP1 expression in BAT cells [68]. Additionally, neurons in the DMH can increase BP, HR, and BAT activity, and, interestingly, DMH neurons can regulate cardiac function and BAT activity at least partially through a relay in the RPa [111,113,115]. This is an interesting finding because sympathetic spinal nerves originating from IML, T1 T4 then innervate both the heart and BAT (Figure 1) [80]. By contrast, the kidney is innervated by IML, T10 T12, and DMH-mediated increase in BP is primary through a relay with the RVLM rather than the RPa [80,113,116]. Hence, neurons in the DMH appear important in the mediation of SNA outflow, although an ability to distinguish between the BAT deposits and the kidney could be present. It will be important to determine the chemical phenotype and projection patterns of lepr-expressing DMH neurons, so that we can understand how this important nucleus contributes to autonomic regulation of thermogenesis and cardiovascular parameters [117]. The extrahypothalamic region: the NTS LepRs are also distributed in additional regions throughout the brain, including the NTS, which is important in cardiovascular control [84]. The NTS can change the glutamatergic input into the RVML and indirectly to the IML, thus altering sympathetic outflow to target organs. Additionally, brain regions that contain leprs (including areas from the hypothalamus) directly innervate the NTS [84]. Leptin acting directly in the NTS decreases food intake and body weight [118,119]. The NTS also participates in leptin-mediated control of SNA. Leptin administration directly into the NTS of lean rats dose-dependently increased rsna, an increase that was specific to the kidney and did not occur in other regions, including BAT [120]. Such findings highlight that extrahypothalamic regions may also be the cause for elevated BP developing in obesity. Other peripheral hormones important in weight control also influencing BP Hormones other than leptin also act to influence SNA and BP, including ghrelin. Ghrelin causes opposite effects to leptin on the melanocortin system: it increases the activity of AgRP/NPY neurons, therefore inhibiting MC4Rs. In rats, peripheral administration of ghrelin caused a dosedependent decrease in HR and BP. Furthermore, ghrelin acting centrally can significantly decrease BP, HR, and rsna [121]. Human studies have confirmed these findings, showing that, at least acutely, ghrelin significantly lowers BP [122]. Insulin can produce actions in the brain and regulate sympathetic outflow via the melanocortin system [89]; it can also increase SNA via the ARH [123]. Plasma insulin levels are generally significantly elevated in obesity. The ability of insulin to increase SNA, especially to skeletal muscle, is of critical importance in stimulating glucose uptake and regulating glucose homeostasis. Infusion of insulin into lean rats increased lumbar, renal, adrenal, and BAT SNA; however, consistent data of insulin increasing SNA, HR, and BP chronically are debatable [124,125]. Hyperinsulinemia is associated with an increase presence of hypertension [126]. However, several animal models that suffer obesity and hyperinsulinemia are normotensive, hence additional 129

research is needed to determine the true role of insulin in the development of hypertension in obesity. Given that leptin and insulin impact the same signaling pathways, specifically the phosphatidylinositol 3-kinase (Pi3K) pathway, increased insulin levels may be involved in worsening the ability of leptin to increase SNA and hypertension. Insulin actions are via the PVH because blockade of PVH neuron activity blocked insulin-mediated changes in SNA. However, insulin does not act directly in the PVN because microinjection into this region produced no change [124]. Thus, insulin is hypothesized to be acting at upstream hypothalamic regions. Other hormones, including glucagon-like peptide-1 (GLP-1), also act to influence SNA, thermogenesis, and BP [127 129]. Hence, peripheral weight-regulating hormones appear critical in the regulation of not only food intake, but also SNA and BP. Concluding remarks Obesity is a key risk factor in the development of hypertension, yet we do not fully understand why it increases the risks of hypertension. Leptin is crucial to the regulation of energy homeostasis and also appears crucial in the maintenance of elevated BP in obesity. Increased activation of the SNS is an appropriate and adaptive response to increased nutrient stores. Activation of the SNS seeks to decrease excessive fat by increasing EE. Unfortunately, this adaptive increase in SNA tone can spillover into other tissue beds, with pathological effects. It might be that sympathetic overactivity is the cause of obesity-related diseases, including hypertension. Treatments for hypertension currently do not target the cause of increased SNA, therefore research must concentrate on understanding the factors, regions, and circuitry that are involved in the elevation of SNA and, hence, elevated BP occurring in obesity. 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