First printed in R&D Systems' 1997 Catalog.
|Figure 1. In the brain, OB contacts OB-R in the choroid plexus. OB is then transported into the CSF where it duffuses into the third ventricle and binds to OB-R on arcuate nucleus (AN) neurons. OB binding to AN parvocellular neurons downregulates NPY levels in these neurons, resulting in an upregulation of autonomic activity in targets such as the PVN. Activated sympathetic activity results in a reduction in appetite and increase in brown fat thermogenesis.
The discovery of leptin (OB, the product of the ob gene) had long been anticipated before its actual isolation. Almost two centuries have passed since it was first proposed that energy balance, in the form of food intake vs. energy output, was physiologically regulated.1 The uncertainty, of course, was where and how such regulation might take place. The hypothalamus was subsequently identified as a key site for body weight regulation. More specifically, the ventromedial nucleus of the hypothalamus (VMH) was found to regulate food intake and energy expenditure, thus influencing overall body weight.2
But the question arose as to how stored energy could be sensed by the VMH, thereby facilitating its continuous adjustment of food intake and energy expenditure. Three theories evolved to explain the general sensing mechanism. One theory proposed that thermoregulation or body temperature influenced the VMH;2 a second theory, termed the glucostatic theory, posited that plasma glucose regulated overall energy stores;3 and a third theory, called the lipostatic theory, suggested there was a product of fat metabolism that circulated in the blood and interacted with the VMH.4 The first two theories failed to adequately explain the precision with which body weight and fat stores were regulated. However, the third theory also suffered due to the absence of an identifiable circulating factor.1 Evidence for such a factor was finally provided by Hervey in 1959.5 At that time, he reported on a parabiosis experiment involving one VMH-lesioned and one VMH-normal rat (Parabiotic animals are animals whose circulatory systems have been joined surgically and who share a common circulation.). Due to damage in the VMH, the lesioned rat exhibited hyperphagia and grew obese. By contrast, the normal rat grew thin, presumably due to reduced appetite. A plausible explanation for these results was that the obese animal produced an increased level of a satiety factor to which it has become insensitive as a result of its VMH lesion, but to which the normal animal responded by eating less and losing weight. The nature of the factor was unknown.5
Although the factors that are now known to influence appetite and energy expenditure are many and complex,6 it would now appear that the proposed "lipostasis theory" circulating factor is none other than the newly discovered molecule termed leptin (OB).1, 7
Human OB is a 16 kDa, 146 amino acid (aa) residue non-glycosylated polypeptide.1, 8 The molecule contains no consensus sites for N-linked glycosylation, but does contain two cysteines in the carboxyterminal region, both of which are believed to participate in an intramolecular disulfide linkage.1, 8 The molecule is translated as a 167 aa residue polypeptide with the first 21 aa residues cleaved as a signal peptide.1 At the amino acid level, human OB is 85% identical to mouse OB and 84% identical to rat OB. Mouse and rat OB exhibit 96% aa identity with each other.1, 9 As might be anticipated based on their homology, both mouse and human OB are active in the mouse system.7 The only cell currently reported to secrete OB is the mature adipocyte.1, 10 Secreted OB has been measured under a variety of conditions and from a variety of sources.11-14
In both human and mouse, the gene for OB is composed of three exons and two introns, and alternative splicing is theoretically possible.15, 16 In particular, there is a possibility that glutamine at position number 49 could undergo deletional splicing. However, to date in the human there is no evidence that this actually occurs.17 In mouse, there is a notable mutation that occurs in the coding sequence at codon number 106 (normally an arginine residue). Here, a cytosine to thymidine change creates a stop codon that causes premature termination of the OB molecule. This mutation disrupts functional OB production and accounts for the abnormalities associated with the ob/ob mouse.1 Although the mouse has documented genetic defects in the OB system that can account for select obese conditions, an equivalent situation does not appear to exist in humans.18 Nevertheless, there does appear to be a genetic predisposition to obesity in some humans, based on the relationship of the OB gene to other genes in the 7q31.3 chromosomal region.19, 20
The receptor for OB has been identified in mouse,21-23 human21 and rat.24 In mouse, the mature receptor is a 1142 aa residue, type I (extracellular N-terminus) transmembrane protein with a predicted molecular weight of 81 kDa. The molecule shows 817 aa residues in its extracellular segment, 21 aa residues in its transmembrane domain, and 302 aa residues in its cytoplasmic tail.21, 23 Mouse, human and rat OB receptors are all virtually identical in length, with the mouse extracellular and cytoplasmic segments exhibiting 77% and 72% aa identity with their human counterparts.21, 23 The OB receptor is described as being a gp130 analog.21, 24 The extracellular region contains Trp-Ser-X-Trp-Ser motifs,21, 24 and the cytoplasmic tail possess both Jak and Stat interaction sequences.21, 22, 25 It would appear that these sequences are utilized as the OB receptor (OB-R) has recently been demonstrated to activate Stat-3, 5 & 6.25, 26
Alternative gene splicing has been extensively documented in the mouse system, with particular emphasis placed on cytoplasmic tail variants. The full length, or long form, is the form noted above with the 300 aa residue cytoplasmic tail. Substantially shorter forms are also known. All of these forms contain the first 29 aa residues of the full length cytoplasmic tail. At this point they vary, adding anywhere from three to eleven aa residues before terminating. A tail with 34 aa residues is designated an OB-Ra form, while receptors with tails of 32 and 40 aa residues are referred to as OB-Rc and OB-Rd forms, respectively.22 Although each of these short tails contains a Jak interaction box,23 there is no evidence that it is functional.26 At least one variant (OB-Re) terminates before the transmembrane segment and this may represent a soluble receptor.22 With respect to the role that OB-R may play in experimental obesity, in fatty Zucker rats a point mutation in the extracellular domain (a glutamine to proline substitution) is suggested to impair receptor dimerization, thus inhibiting signal transduction.27 In the case of the db/db mouse, a predisposition exists for all cells to express alternatively spliced short forms, thus leaving these mice with an apparently non-signaling OB receptor system.22 Anatomical regions suggested to express the soluble OB receptor include adipose tissue stores, the hypothalamus, heart, and testis;22 with short forms associated with the choroid plexus22 and long forms associated with the hypothalamus (arcuate, ventromedial and dorsomedial nuclei) and testis.22, 28 Notably, pancreatic beta-cells that produce insulin have also been identified as expressing OB-Rs, although the form is unknown.29 Regardless of the form of the receptor, however, OB always seems to bind with high affinity (Kd = 250-700 pM).21, 30 Finally, although both mouse and rat have documented defects in receptor expression, to date there is no identifiable functional "aberration" within the human OB receptor system.31
OB was hypothesized to be some sort of "satiety factor", because the absence of this factor, was associated with hyperphagia and obesity in ob/ob mice.32 Following its isolation, it appeared that OB was involved in appetite regulation, due to the fact that OB injections into ob/ob mice reduced their feeding and ultimately their body weight.33, 34 Subsequent studies, however, have demonstrated that its effects must be more complicated than simple appetite suppression. For example, lean mice when injected with OB do lose considerable weight, yet only marginally reduce their appetite.35 And for mice with diet (not genetic)-induced obesity, it takes five-to-ten times more OB to reduce their fat stores relative to weight-matched ob/ob mice.34 In addition, human obesity is often associated with increased blood OB levels,36 suggesting either an insensitivity to OB develops37 or OB's effects are more diverse than the simple description of "satiety factor" would warrant.35, 38
Regardless of the nature of OB's activities, it seems clear (experimentally at least) that OB can be induced (secreted) from white fat cells,39 that it can suppress feeding, and that it can lead to body weight reduction.40 With respect to its release, factors reported to induce OB secretion include insulin39, 41 and inflammatory mediators such as LPS, IL-1 beta and TNF-alpha.42 The effect of insulin is suggested to be part of a negative feedback loop where insulin stimulates OB secretion and circulating OB inhibits insulin production (independently of feeding).38 As noted earlier, OB receptors have been identified on insulin-producing pancreatic beta-cells29 Although speculative, there is evidence to suggest that adipose cell size is a major determinant of OB mRNA expression.43 Perhaps insulin, plus the size of white adipocytes (reflecting stored fat levels), combine to determine the actual quantity of OB released. With respect to the "satiety center", or target of OB action, the hypothalamus has received a great deal of attention. Although the ventromedial hypothalamus was originally considered the OB target, it is now proposed that the hypothalamic arcuate and dorsomedial nuclei are also involved, based on neuroanatomical connections and OB receptor expression.22, 28, 44, 45 It is suggested that circulating OB enters the CNS through the choroid plexus, an area known to express short-form OB-Rs.46 Binding here would not necessarily be associated with signal transduction, only transport across cell membranes. Once in the CNS (perhaps in CSF), arcuate nucleus neuropeptide Y (NPY) neurons could be activated via binding of OB to long-form OB-R.22, 28, 45 This could activate a series of downstream neurons that influence feeding behaviors plus the autonomic nervous system.44 The effect on NPY is not proposed to be facilitatory, however. Increased OB (due to abundant fat stores) decreases NPY expression, an action that activates the sympathetic nervous system and stimulates the heat producing activity of brown adipose tissue (BAT) resulting in increased whole-body energy expenditure and weight loss.44
While the theory is attractive and incorporates known centers involved with metabolism, it has recently been reported that NPY-deficient mice are normal in food intake and body weight and are only induced to over-eat following starvation.47 Thus, alternatives likely exist which can replace the NPY-dependent pathway. In addition, humans, in contrast to rodents, have very small BAT deposits, and it is not clear what role they may play in thermally-based weight loss.48 One suggested alternative role for OB predicts that its true importance lies in its ability to regulate the neuroendocrine system during periods of starvation.49 Obviously more research must be done on all metabolic aspects of OB.
Finally, the OB system has also been proposed to contribute to early hematopoiesis. In mice, a novel hematopoietin receptor found on very primitive hematopoietic cell populations has now been tentatively identified as being an OB-R isoform.50 What role it plays in blood cell expansion and/or development is currently under investigation.
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