1 Introduction: the discovery of leptin—a new perspective for the conception of adipose tissue and the study of obesity
The discovery of leptin in 1994 [
1] represented a key milestone in the study of obesity and the knowledge of the molecular mechanisms involved in body weight control [
2]. Leptin produced by the adipose tissue acts as an afferent signal in a negative feedback loop in the homeostatic control of adipose tissue mass, regulating food intake and energy expenditure [
3]. Moreover, the finding that leptin was produced by the adipose tissue contributed to entail a change in the vision of this tissue. It is now considered as an endocrine organ, whose function goes much beyond that of mere storage of energy reserves, as it has implications in the maintenance of body homeostasis; this rekindled interest in this tissue [
4]. In short, the discovery of leptin opened up a new molecular perspective in the conception of the adipose tissue and in the approach to the study and treatment of obesity, including new therapeutic targets, involving leptin itself, its regulation, and post-leptin processes downstream of leptin.
Leptin was initially described as an anti-obesity hormone. The lack of leptin in ob/ob mice was associated with early-onset, morbid obesity [
1]. Leptin administration was found to restore normal body weight and reversed other alterations related to the absence of leptin, including hyperphagia, low body temperature, decreased metabolic rate, immunodeficiency, and insulin resistance [
5‐
7]. Similar phenotypic abnormalities were found in humans with congenital leptin deficiency [
8], and leptin administration was shown to effectively treat obesity, with the most significant impact being mediated by its suppressive effects on food intake [
9]. But leptin protects not only against obesity, it really serves as an adiposity signal, with a critical function in maintaining adipose tissue mass to ensure survival in conditions of negative energy balance, thereby protecting individuals from the associated risks of having both a deficit and an excess of adiposity [
3]. Nevertheless, human obesity is not generally caused by leptin deficiency, neither mutations of the leptin gene or the leptin receptor gene are frequent in humans, but leptin levels are commonly higher in subjects with obesity, and insensitivity to endogenous leptin is a hallmark of most cases of human obesity [
10].
Although adipose tissue is the primary producer of leptin, other tissues also produce it, such as the stomach [
11,
12] ̵ with a main function in the short-term regulation of food intake [
13‐
15] ̵ as well as mammary gland [
16] and placenta [
17] ̵ with long-lasting effects in development [
18]. Furthermore, we now know that leptin is involved in many other functions besides body weight control, including reproduction, glucose homeostasis, hematopoiesis, and immune function [
19,
20]. Moreover, leptin plays essential roles during critical periods of development in foetal and infant development and as a potential programming factor [
18], therefore exerting short, medium and long term actions, broadening the view of leptin as a pleiotropic hormone. For it, leptin exerts regulatory functions in different tissues, including the adipose tissue itself, and the action in this tissue represents a fundamental target in its main role in maintaining energy homeostasis. Comprehending the effects of leptin on adipose tissue and its regulation is critical to better understand the pathophysiology of obesity and related diseases, such as type 2 diabetes, as well as their prevention and treatment. Here, we aim to provide a comprehensive review of the main regulatory effects of leptin on adipose tissue under physiological and pathological conditions, such as obesity, the mechanisms and pathways involved, its influence in critical windows of development, and its long-lasting effects on metabolism and health.
2 The endocrine adipose organ: leptin production and regulation
The adipose organ constitutes the main energy reservoir of the organism, which is the main place to accumulate the energy surplus as triglycerides (TG). This definition is undoubtedly an outdated view of the role of the adipose organ in metabolic homeostasis. Now, the pleiotropic functions of this organ are evident, among them being an endocrine organ that regulates metabolic pathways, which depends mainly on the type of cells and the location of the tissue [
21,
22].
The adipose organ can be divided into two different main types of depots: white adipose tissue (WAT) depots, and brown adipose tissue (BAT) depots, with different functions and morphology [
23]. Both types can be divided into various depots depending on their localization: subcutaneous, visceral, periaortic, etc., which exhibit different cellular composition, secretory capacity, vascularization, and innervation [
24]. On the one hand, white adipocytes are unilocular cells, presenting one big fat vacuole that represents around 90% of the cell volume, where fatty acids (FA) are stored mainly as TG, which can be mobilized and distributed to different tissues when needed [
23]. On the other hand, brown adipocytes are multilocular and rich in mitochondria, containing the uncoupling protein 1 (UCP1), a protein responsible for adaptive non-shivering thermogenesis, by uncoupling of the oxidative phosphorylation from ATP synthesis, exerting a key role on heat maintenance, particularly in newborns and hibernating mammals [
25,
26]. Both types of cells are able to carry out the storage and mobilization of TG in response to the organism demands. Although the adipocyte is the characteristic cell, the adipose tissue also comprises other cell types such as stem cells, preadipocytes, fibroblasts, stromal cells, T-cells, granulocytes, macrophages and monocytes [
24].
WAT displays a key role as an endocrine organ metabolically active by secreting a variety of functional products, called adipokines, which can act in autocrine, paracrine, or endocrine manners, establishing complex cross-talk between adipose tissue and other organs [
24]. Probably, the so far considered most relevant adipokine is leptin, which is predominantly secreted from visceral fat in rodents [
27] and from subcutaneous fat in humans [
28]. The adipocytes secrete leptin into the bloodstream proportionally to fat mass; hence leptin levels are strongly correlated with adiposity in rodents and humans, serving as an adiposity signal [
29].
Leptin expression and protein levels in the adipose tissue are regulated by many factors, including a variety of hormones, such as insulin [
30], glucocorticoids [
31,
32] and catecholamines [
33], and show diurnal variations, according to the nutritional status [
34]. In humans, leptin levels are high at night and low around noon and early afternoon period [
35]. However, these changes are relatively modest (about 1.5-fold), compared to those associated with metabolic and nutritional states, which determine rapid changes in leptin synthesis regardless of changes in the size of fat depots [
36]. Circulating leptin levels respond to nutritional state or feeding patterns, rising after feeding, when insulin levels are increased, and decreasing under fasting conditions or weight loss [
29,
33,
37].
In vitro and
in vivo studies support the direct role of insulin in leptin production. In starved rats, the combination of insulin administration and refeeding has been described to increase leptin mRNA expression to a similar extent to insulin alone, indicating that insulin is sufficient to mimic the effects of food intake on leptin expression [
30]. Glucocorticoids also upregulate leptin mRNA expression, and chronic exposure to insulin further enhances leptin release [
32]. In fact, the combination of hyperinsulinemia and the increased cortisol turnover that is associated with the obese state has been proposed to contribute to the maintenance of hyperleptinemia in obese individuals [
38]. Conversely, the activation of the sympathetic nervous system via catecholamines contributes to a rapid decline in circulating leptin levels, therefore providing a mechanism in response to fasting and cold exposure [
39,
40]. On the contrary, sympathetic blockade increases leptin gene expression and circulating leptin levels [
41]. The fact that the sympathetic sensitivity of adipose tissue is reduced in obesity also contributes to explain the presence of increased leptin levels in the obese state, aside from the limited cases where leptin is absent [
42].
The regulatory mechanisms involved in the transcriptional control of the leptin gene are poorly understood. Dallner et al. [
43] have recently identified a fat-specific long non-coding RNA (lncOb), which is regulated in concert with fat mass and that regulates leptin expression by interacting with redundant enhancers. Lack of functional
IncOb in mice is associated with decreased plasma leptin levels and increased adiposity, but these animals are responsive to leptin treatment. In humans, certain single-nucleotide polymorphisms in this locus are associated with decreased circulating leptin levels and obesity [
43]. These findings seem clinically relevant regarding leptin therapy, particularly for the subset of patients with obesity who have relatively low circulating levels of leptin.
3 Leptin action and leptin resistance
Leptin acts through its specific leptin receptor (LEPR), which exhibits a widespread distribution in the central nervous system (CNS) and peripheral organs. LEPR is encoded by the
LEPR (or
OBR) gene and belongs to the cytokine class 1 family [
44]. Six isoforms have been identified, which have a common leptin-binding domain but differ in the length of C-terminal intracellular domain [
45]. These isoforms include the long form (LEPRb) that contains the intracellular motifs required for full activation of the signalling pathways upon leptin binding, four short forms (LEPRa, LEPRc, LEPRd, and LEPRf), and one soluble form (LEPRe) without the transmembrane and cytoplasmic regions [
46]. The reasons why distinct forms of leptin receptors are produced and their respective functions are not yet clear. The expression and characteristics of the leptin receptors isoforms, and the complete pathway of leptin signalling through LEPRb activation, can be revised elsewhere [
46].
The principal neuronal targets of leptin are located in specific areas of the hypothalamus, a brain region with a key role in the control of feeding and energy expenditure [
47]. The best-known leptin-sensitive neuronal systems start from the arcuate nucleus (ARC) of the hypothalamus, which contains neurons rich in the leptin receptor. Two main types of neurons with antagonistic functions are major targets of leptin. Leptin stimulates pro-opiomelanocortin (POMC) neurons responsible for the synthesis of POMC and cocaine‐ and amphetamine‐regulated transcript (CART), which are anorexigenic neuropeptides [
48]. POMC is processed to form α-melanocyte-stimulating hormone (α-MSH), which is released from POMC axon terminals and activates melanocortin 3 and 4 receptors on downstream neurons [
48]. Conversely, leptin acts on agouti-related peptide (AgRP) neurons in the ARC nucleus and inhibits the synthesis of the orexigenic AgRP and neuropeptide Y (NPY) neuropeptides, which may have complementary functions [
49]. Both groups of neurons send projections to other hypothalamic nuclei involved in the control of energy balance, including the paraventricular nucleus (PVN) and the lateral hypothalamic area (LHA) [
2]. Besides central action, leptin exerts a diverse range of peripheral actions related to its main roles in the regulation of energy balance [
50,
51].
Leptin produced by the adipose tissue is not viewed primarily as a short-term satiety signal since circulating leptin levels may take several hours to change after food consumption [
36]. However, leptin produced by the stomach may take part in the short-term control of food intake, together with other satiety signals [
12,
15]. Leptin produced by the stomach is secreted to the gastric lumen and general circulation [
12]. The amount of leptin released to circulation is probably not large enough to produce systemic effects as leptin produced by the adipose tissue does; rather, gastric leptin may act locally, and provide rapid information to the brain via peripheral vagal afferent pathways that originate in the stomach and intestine and terminate in the nucleus tractus solitarius, inducing satiety [
15].
According to the anorexigenic role of leptin, exogenous leptin therapy reduces food intake and promotes weight loss in lean animals or humans with low circulating leptin levels [
52‐
54]. In patients with congenital leptin deficiency, leptin is able to normalize energy balance, through a major effect on the reduction of food intake [
55,
56]. Equally true is that most subjects with obesity are not deficient in leptin, rather exhibit higher circulating leptin levels than those in non-obese subjects, which is a feature of leptin resistance [
10]. Subjects with generalized lipodystrophy and low leptin levels may also benefit from the administration of recombinant leptin as replacement therapy to manage metabolic diseases and comorbidities, since it has been reported to improve hypertriglyceridemia, glycaemic control, and overall liver health [
57], and potentially reduce the risk of mortality [
58].
Leptin resistance refers to the condition in which the brain or peripheral tissues are less sensitive (or do not respond) to leptin, and therefore leptin fails to promote its anticipated effects [
59,
60]. This state could result in a vicious cycle, as it leads to a further increase in circulating leptin levels, and therefore to a worsening of leptin resistance. Therefore, leptin itself plays an important role in the development of its resistance [
61]. The underlying mechanisms of leptin resistance are not fully clarified although, to date, several mechanisms have been proposed related to an impairment in leptin transport to the brain across the blood–brain barrier (BBB), or others alterations downstream of the leptin receptor, which are briefly discussed below (reviewed in [
60]).
Leptin transport into the brain seems to be a limiting step in its central effects, but the way how blood leptin gains access to its target neurons in the brain is still an open question [
60]. In subjects with obesity, elevated leptin levels in blood are not paired by proportionally high leptin levels in the cerebrospinal fluid, suggesting a causal relationship between a deficit in the transport system that carries leptin to the CNS and obesity [
62]. Studies in obese mice show that these animals are sensitive to central administration of leptin but not to subcutaneous or intraperitoneal administration, indicating that the lack of leptin effect could be due to impaired transport of blood leptin to the brain [
63]. However, the contribution of the alteration of leptin transport through the BBB to leptin resistance is not clear. Some studies have also shown that leptin resistant mice maintain intact transport of leptin through the BBB [
64,
65], thereby its real contribution as a mechanism of leptin resistance has been questioned. Other studies in animal models have suggested that the impairment in the brain leptin transport is acquired rather than causal since it is developed secondary to obesity and may be reversible with weight reduction [
66]. Therefore, although it is unlikely that alterations in the leptin transport through the BBB are directly involved in the development of leptin resistance, we cannot rule out that defects in this mechanism could play a role [
20].
Of interest, recent studies have pointed out the role of tanycytes - a specialized glial cell type that line the third ventricle in the median eminence of the hypothalamus - in the shuttle of leptin and other hormones into the cerebrospinal fluid, also playing a potential role in the pathophysiology of central leptin resistance [
67,
68]. In certain brain structures called the circumventricular organs, characterized by capillaries harbouring a fenestrated endothelium, the BBB consists of tanycytes, which extend from the wall of the cerebral ventricles and these fenestrated capillaries. These cells have barrier properties, preventing the diffusion of circulating molecules from fenestrated capillaries to the rest of the brain via the cerebrospinal fluid (see [
68]). Tanycytes are possibly the first cells in the hypothalamus that respond to circulating leptin. They can take up leptin that freely exits the hypothalamic-pituitary portal blood circulation through fenestrated capillaries, and shuttle it into the brain to reach their target neuronal populations in an extracellular signal–regulated kinase (ERK)-dependent manner [
67]. The transport of leptin seems to require the activation of a leptin receptor-epidermal growth factor receptor (LEPR–EGFR) complex [
69]. Tanycytes appear to be the first cells of the brain to become resistant to leptin in diet-induced obesity, and this alteration is prior to the appearance of metabolic dysfunctions [
68]. Interestingly, EGF treatment in diet-induced obese mice has been described to activate ERK in tanycytes, re-establish leptin transport, and restore the capacity of exogenous peripheral leptin to activate signal transducer and activator of transcription 3 (STAT3) in hypothalamic leptin-sensitive neurons [
67].
Changes in the leptin receptor expression could also affect leptin sensitivity [
70]. Hypothalamic
Lepr mRNA (long form) downregulation has been found in animal models displaying hyperleptinaemia [
71]. Interestingly, mechanisms leading to decreased
Lepr mRNA expression may be early programmed [
72]. Several studies have shown an association between the metabolic programming of obesity by adverse conditions during gestation and the presence of lower
Lepr mRNA levels in the hypothalamus (often accompanied by a decrease in the expression of the insulin receptor), as well as an alteration in leptin signalling [
73‐
76]. As an example, moderate calorie restriction during gestation in rats has been shown to be associated with higher food intake and higher body weight and adiposity in adulthood, in a sex-dependent manner, and this has been attributed, in part, to a diminished response to leptin action in adulthood [
76,
77]. Notably, these animals already displayed lower
Lepr mRNA levels in the hypothalamus at early stages of life (after weaning), suggesting central leptin resistance as an early adverse effect of gestational undernutrition, which may be responsible for later energy homeostasis dysregulation [
76].
Overexpression of negative regulators of leptin action, such as the suppressor of cytokine signalling 3 (SOCS3) [
78], and the tyrosine phosphatases (PTPs) tyrosine phosphatase 1 B (PTB1B) and T cell protein tyrosine phosphatase (TCPTP) [
79], may also contribute to leptin resistance. Mice with haploinsufficiency of
Socs3 display greater leptin sensitivity than wild-type mice and are protected against the development of diet-induced obesity [
80]. In turn, hypothalamic expression of PTPs has been found to be increased in obese animals, and selective ablation of these proteins partially prevents diet-induced obesity and improves leptin sensitivity [
81‐
83], providing a demonstration of their role in the onset of leptin resistance. Moreover, and possibly in connection with the key role of SOCS3 and PTP1B in leptin action, endoplasmic reticulum (ER) stress has also been identified as a mechanism implicated in the development of obesity-associated leptin resistance [
60,
84,
85]. ER stress may be the result of an excessive accumulation of unfolded proteins, activating the unfolded protein response (UPR) [
86]. There is evidence that leptin resistance may be mediated, at least in part, by SOCS3 and PTP1B [
60]. The expression of these proteins is inhibited by X-box binding protein 1 (XBP1), a main regulator of ER folding capacity [
60]. Pharmacological induction of ER stress or deletion of XBP1 in neurons result in hyperleptinemia and obesity, associated with severe hypothalamic leptin resistance [
84]. Conversely, selective overexpression of XBP1 in POMC neurons prevents the ER stress-mediated blockade of LEPRb and restores leptin sensitivity [
84,
87].
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