PI3K signaling: A key pathway in the control of sympathetic traffic and arterial pressure by leptin

Abstract

The adipocyte-derived hormone, leptin, is a master regulator of energy homeostasis. Leptin action in the central nervous system also contributes to arterial pressure regulation through its capacity to increase renal sympathetic outflow. The accumulating evidence pointing to a key role for leptin in the adverse sympathetic and cardiovascular consequences of excessive adiposity highlight the importance of understanding the mechanisms underlying the sympathetic and cardiovascular effects of leptin. The ability of the leptin receptor to stimulate various intracellular pathways allows leptin to regulate physiological processes in a specific manner. In this review, we examine the role of the PI3K pathway emanating from the leptin receptor in mediating the sympathetic and arterial pressure effects of leptin. We also discuss the relevance of PI3K signaling for obesity-induced hypertension through its role in mediating selective leptin resistance.

1. Introduction

The importance of the central nervous system (CNS) in maintaining homeostasis is now well recognized. The CNS influence on physiological processes occurs through efferent humoral and neural signals. For this, the brain controls the release of key hormones and the activity of the sympathetic and parasympathetic branches of the autonomic nervous system. In turn, the brain receives a myriad of information from peripheral tissues about their status. The adipocyte-derived hormone, leptin, is an example of the afferent input that informs the CNS about the status of body’s energy reserves [1]. Plasma levels of leptin are proportional to fat stores and changes in circulating leptin levels are detected by the CNS which then adjusts energy intake and expenditure, the later mediated through the sympathetic nervous system [2]. Indeed, CNS action of leptin increases sympathetic activity of the nerves subserving various tissues involved in metabolic control such as brown adipose tissue (BAT), white fat pads, liver and skeletal muscle [3,4].

Besides its critical role in the control of energy homeostasis, leptin action in the CNS is implicated in the regulation of a number of physiological functions including glucose metabolism, sexual maturation and reproduction, the hypothalamic–pituitary–adrenal system, thyroid and growth hormone axes, angiogenesis and lipolysis, hematopoiesis, immune or pro-inflammatory responses, and bone remodeling [5–9]. Leptin also contributes to the regulation of arterial pressure with important pathophysiological implications in obesity-associated hypertension [3]. Leptin impacts arterial pressure through its ability to increase sympathetic nerve activity subserving cardiovascular organs such as the kidney. In addition, it is well established that leptin resistance, particularly with respect to its anorexigenic action, contributes to obesity development [2,9]. However, leptin’s sympathetic/cardiovascular effects are preserved under obese conditions. This selectivity in leptin resistance appears to contribute to the obesity-associated sympathetic overdrive and hypertension [10,11]. In this review, we will discuss the molecular pathways triggered by leptin binding to its receptor and the importance of the PI3K signaling in mediating the sympathetic and cardiovascular actions of leptin in health and disease.

2. Leptin receptor (LepR) signaling

The leptin receptor belongs to the class 1 cytokine receptor family [12]. Despite the existence of many splice variants of the leptin receptor, the long LepRb isoform which is lacking in db/db mice is the most relevant for the physiological actions of leptin. A large number of signaling pathways have been associated with the LepRb. In the CNS, leptin binding to LepRb leads to the activation of at least four major signaling pathways. These four signaling pathways have been shown to be involved in mediating the effects of leptin on energy homeostasis (Figure 1).

Figure 1.

Intracellular signaling pathways regulated by the leptin receptor (LepRb) in the central nervous system. Four primary intracellular signaling pathways emanates from LepRb: signal transducer and activator of transcription (STAT) 3 and STAT5 proteins, the extracellular signal-related kinase (ERK) and phosphoinositol-3 kinase (PI3K). Each pathway is activated by phosphorylation of specific tyrosine residues in ObRb. The four signaling pathways are implicated in leptin regulation of energy homeostasis. In contrast, leptin control of renal sympathetic nerve activity (SNA) and arterial pressure occur through PI3K signaling.

Binding of leptin to LepRb triggers a conformational change in the receptor which promotes its activation by autophosphorylation through Janus kinase (Jak) 2 tyrosine kinase. Activated Jak2 then phosphorylates three tyrosine residues within the LepRb at position 1138, 1077 and 985. Each of these tyrosine residues mediates the activation of distinct downstream signaling pathways [13,14]. Phosphorylated Tyr₁₁₃₈ promotes the recruitment and phosphorylation of signal transducer and activator of transcription (STAT) 3, whereas phosphorylated Tyr₁₀₇₇ binds and phosphorylate STAT5 [15]. Activated STAT3 and STAT5 translocate to the nucleus to modulate gene transcriptional with implications for physiologic regulation [16]. Interestingly, these two transcription factors appear to be differentially engaged by leptin to control physiological processes. Indeed, mice carrying a mutation at Tyr₁₁₃₈ of ObRb, which disrupts leptin-induced STAT3 activation, are severely obese and hyperphagic, but in contrast to the mice lacking leptin or LepRb these mice remain fertile and are less diabetic [6]. Conversely, knock-in mice bearing a mutation at Tyr₁₀₇₇ of LepRb, which disrupts leptin-induced STAT5 signaling were found to have impaired reproductive function, but exhibited only a modest increase in adiposity [58].

The phosphorylated Tyr₉₈₅ of LepRb leads to the recruitment and binding of the COOH-terminal SH2 domain of the tyrosine phosphatase, SHP2, leading to the activation of extracellular signal-related kinase (ERK) pathway [13,14,17,18]. Tyrosine phosphorylation sites on Jak2 also appears to contribute to leptin-induced ERK activation independent from LepRb phosphorylation [13]. Tyr₉₈₅ (and perhaps Tyr₁₀₇₇) also binds suppressor of cytokine signaling-3 (SOCS3) which act as a negative regulator to inhibit STAT3 signaling. Protein tyrosine phosphatase 1B is another negative regulator of LepRb through a direct action to dephosphorylate Jak2 [9,19].

Stimulation of LepRb also promotes the activation of phosphatidylinositol-3 kinase (PI3K) pathway. A role for the PI3K pathway in transducing leptin action was initially identified in vitro using various cell types and confirmed later in vivo. For instance, in C₂C₁₂ myotubules, leptin stimulation of glucose transport was shown to be dependent on PI3K pathway [20]. In primary rat hepatocytes leptin was also found to activate PI3K [21]. Moreover, in CRI-G1 insulinoma cells, modulation of K_ATP channels activity by leptin was demonstrated to require PI3K signaling [22]. In hypothalamic brain slices, leptin evoked depolarization of proopiomelanocortin (POMC) neurons in a PI3K-dependent manner [23]. Thus, PI3K signaling has been implicated in mediating leptin action in various cell types. The exact mechanism by which LepRb activates PI3K signaling remains unclear, although this appears to involve the insulin receptor substrates [14].

Several downstream mechanisms that would explain the involvement of PI3K signaling in mediating the metabolic effects of leptin have been suggested, including its activation of phosphodiesterase-3B which can then interact with STAT3 [24] or through inactivation of FoxO-1 transcription factor that stimulate POMC neurons while inhibiting Agouti-related peptide (AgRP) neurons [25,26].

3. PI3K signaling and leptin regulation of energy balance

A number of studies have demonstrated the relevance of PI3K as an underlying mechanism of leptin actions in vivo [24,27]. In rats, peripheral leptin administration was found to activate PI3K in the hypothalamus, a region of the brain known to be critical for the metabolic effects of leptin [27]. Moreover, pre-treatment with inhibitors of PI3K abolished the anorectic response induced by leptin [27,28].

The use of genetically modified mouse models has been critical in establishing the importance of PI3K signaling in the long-term regulation of energy homeostasis and in mediating the metabolic effects of leptin. In addition, the genetically engineered mouse models allowed the identification of the neuronal populations and brain regions where PI3K mediates leptin effects. For instance, a conditional knockout mouse model in which PI3K activity was enhanced specifically in LepRb-expressing cells exhibited increased energy expenditure leading to less adiposity provided strong evidence regarding the metabolic consequences of chronic enhancement of PI3K in LepRb-containing neurons [25]. Other studies used loss of function approach to assess the importance of PI3K signaling in specific neuronal populations of the hypothalamus. Such approach revealed that targeted disruption of PI3K signaling in POMC neurons altered the ability of leptin to decrease food intake in mice [23]. On the other hand, disrupting PI3K in the AgRP neurons rendered the mice leptin-sensitive, lean and resistant to diet-induced obesity [29]. Together, these findings demonstrate that interference with PI3K pathway in a subset of hypothalamic arcuate nucleus neurons alters the ability of leptin to regulate food intake and energy homeostasis. These obsertvations are also consistent with the differential effects of leptin on PI3K activity in POMC and AgRP neurons. Indeed, in POMC neurons leptin stimulate PI3K activity whereas leptin action in AgRP neurons inhibits PI3K activity [30].

A recent study demonstrated the importance of PI3K signaling in the ventromedial hypothalamic nucleus and its involvement in the long-term effects of leptin on energy homeostasis. Indeed, mice with specific reduction in PI3K activity in the ventromedial hypothalamic neurons exhibited resistance to the anorectic effects of leptin associated with lower energy expenditure and enhanced sensitivity to diet-induced obesity [31]. Thus, experimental evidence using a combination of pharmacological approaches and genetically modified mouse models clearly identifies a role for LepRb-PI3K signaling axis in the regulation of energy homeostasis.

4. PI3K signaling mediates the sympathetic and cardiovascular effects of leptin

Numerous studies have implicated leptin in the regulation of arterial pressure [32,33]. For example, the leptin deficient ob/ob mice exhibit reduced arterial pressure as compared to their lean wild type controls despite being massively obese [34]. On the other hand, chronic infusion of leptin or transgenic overexpression of leptin elevated arterial pressure in spite of body weight reduction [35,36]. Importantly, adrenergic blockade reverses leptin-induced arterial pressure increase highlighting the importance of the sympathetic nervous system in mediating the pressor effect of leptin [37].

The role of the various signaling pathways emanating from the LepRb in the transduction of the regional sympathetic activation caused by leptin has been investigated. Interestingly, despite the divergent capacity of the LepRb, the PI3K signaling has emerged as the main pathway linking leptin to sympathetic and cardiovascular function (Figure 1). In mice, PI3K blockade with either LY294002 or Wortmannin markedly attenuated the leptin-induced increase in renal sympathetic activity [28]. These inhibitors did not affect the renal sympathetic activation caused by other stimuli such as MTII, an agonist of the melanocortin 3/4 receptors, suggesting that their inhibitory effect on the response evoked by leptin was specific. In a subsequent study in rat, we found that the role of PI3K in mediating leptin-induced sympathetic activation was specific to the kidney, as pre-treatment with LY294002 prevented the effect of leptin on renal sympathetic traffic without altering the sympathetic responses to other beds including BAT, hindlimb and adrenal gland [38].

The role of STAT3 in mediating the regional sympathetic responses evoked by leptin was assessed using the s/s mouse model. Specific disruption of the LepRb-STAT3 pathway in s/s mice did not alter leptin-induced renal sympathetic activation which seems to exclude a role for STAT3 signaling in the control of renal sympathetic traffic by leptin. However and consistent with their obesity phenotype, the s/s mice exhibited a substantially attenuated leptin-induced BAT sympathetic nerve response [39,40].

The l/l mouse model bearing a selective disruption of LepRb-ERK axis was used to examine the involvement of ERK signaling in the regional sympathetic effects of leptin [41]. The l/l mice exhibited enhanced sensitivity of renal sympathetic response to leptin [40]. Importantly, the increased sympathetic nerve response to leptin in the l/l mice was selective as indicated by the fact that leptin-induced increases in renal sympathetic traffic is enhanced whereas the increase in BAT sympathetic activity evoked by leptin was attenuated [40]. Pharmacological inhibition of ERK signaling also prevented the effects of leptin on sympathetic nerve traffic to thermogenic BAT, but not to the kidney, hindlimb or adrenal gland [38]. The demonstration that PI3K inhibition blocks the renal sympathetic response to leptin in the l/l mice confirms the importance of this pathway in the transduction of leptin-induced renal sympathetic activation [40].

The enhanced renal sympathetic response to leptin in l/l mice translates into a greater arterial pressure increase after leptin treatment [40]. In addition, despite normal body weight, the l/l mice have elevated arterial pressure, heart rate and urinary norepinephrine levels [7,40]. The importance of the sympathetic transmission in the elevated arterial pressure in l/l mice is demonstrated by the augmented depressor response to ganglionic blockade in these animals [40]. These findings demonstrate that selective loss of LepRb-ERK signaling caused by disruption of Tyr₉₈₅ enhances the cardiovascular and renal sympathetic effects of leptin leading to elevated baseline arterial pressure. The fact that STAT3 and ERK pathways are not involved in the renal SNA response to leptin support further the notion that PI3K signaling is the major pathway for the transduction of leptin-induced changes in renal sympathetic outflow and arterial pressure (Figure 1).

Additional evidence implicating PI3K signaling in the sympatheic and hemodynamic action of leptin derives from our recent findings using two contrasting mouse models with either abolished (p110α^D933A/WT mice) or enhanced (Pten^∆LepRb mice) effect of leptin on PI3K signaling [42]. The p110α^D933A/WT mice carry a knock-in mutation that abrogates p110α kinase activity [43] while the Pten^∆LepRb mice have the lipid phosphatase Pten (phosphatase and tensin homolog) ablated in cells expressing the LepRb, leading to enhanced PI3K pathway specifically in leptin-sensitive neurons [25]. Relative to control mice, the Pten^∆LepRb mice exhibited enhanced renal sympathetic and arterial pressure responses to leptin. In contrast, the p110α^D933A/WT mice had blunted sympathetic and hemodynamic responses to leptin. Notably, baseline arterial pressure was elevated in Pten^∆LepRb mice compared to controls whereas the p110α^D933A/WT mice were normotensive [42]. These observations demonstrate the importance of PI3K pathway for the sympathetic and cardiovascular regulation by leptin.

5. PI3K signaling is implicated in leptin-induced sympathetic activation in obesity

Hypertension and sympathetic overdrive are common features of obesity in humans [44–46]. Analysis of regional sympathetic activity revealed a significantly elevated renal sympathetic tone in the obese subjects relative to lean controls [47]. The importance of the renal nerves in preventing obesity-induced hypertension has been demonstrated using bilateral renal denervation in dogs [48,49]. Of note, renal denervation was also found to be efficient in reversing the hypertension after its establishment in obese dogs [47].

Leptin action in the CNS has emerged as a primary culprit in driving the hypertension and the increase in sympathetic discharge commonly associated with obesity [10,32]. In support of this concept are the studies showing a correlation between circulating leptin levels with hypertension as well as renal sympathetic overdrive in obese humans and animal models of obesity [50]. Animal studies provided important mechanistic insights into the role of leptin in obesity-associated cardiovascular side effects by demonstrating that the ability of leptin to increase renal SNA and arterial pressure remains intact in several animal models of obesity including diet-induced obese mice and rabbits despite the blunted appetite- and weight-reducing effects of leptin [51–56].

The evidence implicating hyperleptinemia in obesity-induced hypertension prompted the search for the mechanisms underlying leptin-induced increase in renal sympathetic outflow and arterial pressure in obesity despite the resistance to its metabolic actions. One possibility is that selectivity in leptin resistance may be due to the inability of leptin to activate downstream signaling pathways in the arcuate nucleus, but preservation of leptin actions in other cardiovascular-related brain areas. However, we found that the LepR in the arcuate nucleus mediate the preserved leptin-induced renal sympathetic activation highlighting the importance of this nucleus for the sympathetic effects of leptin in obesity [57]. In addition, LepR in the arcuate nucleus is critical for the elevated arterial pressure in diet-induced obese mice [57]. These findings indicate that, in obesity, leptin receptor-containing neurons in the arcuate nucleus are not uniformly resistant to leptin. The arcuate nucleus neurons that are engaged by leptin to increase renal sympathetic traffic and arterial pressure appear to escape leptin resistance. In addition, there is growing evidence implicating hypothalamic nuclei other than the arcuate nucleus as well as extra-hypothalamic nuclei in leptin regulation of physiological processes including regional sympathetic nerve traffic [4]. However, the relevance of these nuclei to the sympathetic and cardiovascular actions of leptin in obesity remains to be determined.

Another major progress in understanding the mechanisms of selective leptin resistance and cardiovascular side effects of leptin relate to the identification of the importance of PI3K pathway in mediating the preserved renal SNA response to leptin in obesity. Indeed, pre-treatment with pharmacological inhibitors of PI3K attenuated leptin-induced renal sympathetic nerve activation in two mouse models of obesity, DIO and the agouti obese mice [55]. These results demonstrate the pivotal role of the PI3K pathway in the preserved renal SNA response to leptin in obesity and strongly implicate PI3K signaling in mediating the arterial pressure effects of leptin in obesity.

6. Conclusion

Several intracellular pathways can be activated by leptin through various tyrosine residues of the leptin receptor. Such divergent signaling capacities of the leptin receptor are critical for leptin regulation of various physiological functions [16,32]. The studies summarized in this brief review points to the PI3K signaling emanating from the leptin receptor in the CNS as a key pathway underlying the renal sympathetic and cardiovascular effects of leptin with important pathophysiological implications in obesity. However, little is known about the mechanisms downstream to PI3K that are engaged by leptin to control sympathetic and hemodynamic function. Detailed understanding of the precise molecular mechanisms and signaling pathways in the CNS that leptin employs to alter renal sympathetic traffic and arterial pressure will greatly enhance our understanding of the obesity-associated cardiovascular disorders and enable the development of safe therapies.

Conflict of interest

None declared.