ACTION OF LEPTIN ON BONE AND ITS RELATIONSHIP TO MENOPAUSE

BACKGROUND
Leptin a cytokine protein secreted by adipose tissue raises considerable interest as a potential mediator of the protective effects of fat mass on bone tissue. After menopause heavier women conserve bone mass better than those with lower body weight. The protective effect of obesity on bone mass has been ascribed to a high body fat content. As Leptin levels reflect the body fat content it has emerged as a possible mediator of these protective effects.


METHODS
A search of the available literature focused on the role of leptin on bone tissue.


RESULTS
Both peripheral and central action of leptin on bone metabolism have been proposed. In vitro and in vivo evidence supports the hypothesis that leptin can act directly or indirectly on bone remodelling by modulating both osteoblast and osteoclast activities. However, studies in humans have not yet been able to confirm these actions possibly because of the shifting balance between stimulatory direct action and suppressive indirect action of leptin on bones via the hypothalamus. The effects of oestrogen decline and deficiency during natural or artificially induced menopause and administration of hormone replacement therapy has on leptin production remains controversial. Various studies have shown differences in leptin values in pre- and postmenopausal women. The existing clinical data on this issue are discordant.


CONCLUSION
Larger clinical studies are necessary to clarify leptin's role in vivo and to assess the contribution of the central and peripheral role of leptin in the overall maintenance of bone turnover in human beings.


INTRODUCTION
Leptin, a member of the helical cytokine family is the soluble 16-kDa product of the OB (Lep) (Ob for obese and Lep for leptin) gene on human chromosome 7q31.3and chromosome 6 in mouse 1 .Leptin is mainly produced by white fat cells, it is also produced by brown adipose tissue, placenta (syncytiotrophoblasts), ovaries, skeletal muscle, stomach (lower part of fundic glands), mammary epithelial cells, bone marrow, pituitary and liver 1 .Leptin fi rst emerged as a component of a regulatory loop linking fat mass to food intake and energy expenditure but later also raised considerable interest as a potential mediator of the protective eff ects of fat mass on bone tissue 2 .The protective eff ect of obesity on bone mass has been ascribed to a high body fat content.Leptin is produced by adipose cells, with its levels refl ecting the amount of adipose tissue, so leptin logically emerged as a possible mediator of protectice eff ect of fat tissue on bone.Leptin exerts its eff ects through interaction with six types of receptors.Leptin receptors are divided into secretory (ObRe), long (ObRb) and short forms (ObRa, ObRc, ObRd, ObRf).(ObRe) acts as a binding protein for leptin in the plasma in humans and mice and is important for leptin transfer into the brain.ObRb is the only receptor isoform that contains active intracellular signaling domains.This receptor is found in a number of hypothalamic nuclei where it exerts its eff ects.ObRb receptors are also found in other cells including osteoblasts.Leptin binds to the Ventral Medial nucleus of the hypothalamus, known as the "satiety center" where it signals to the brain a sensa-tion of satiety.Leptin also works by inhibiting the activity of neurons that contai the neuropeptide Y (NPY) and agouti-related peptide (AgRP), and stimulating neurons expressing α-melanocyte-stimulating hormone (α-MSH).The NPY neurons are a key element in the regulation of appetite.Once leptin has bound to the Ob-Rb receptor, it activates the stst 3 molecule, which is then phosphorylated and travels to the nucleus,where it is presumed, to eff ect changes in gene expression.One of the main eff ects on gene expression is the down-regulation of the expression of the endocannabinoids, responsible inter alia for increasing appetite.There are several other intracellular pathways activated by leptin, but less is known about how they function in this system.

Mechanism of leptin action on bone
The exact infl uence of leptin on bone metabolism has not yet been clarifi ed.The literature is inconsistent and confl icting.Leptin is emerging as central in two entirely diff erent bone -controlling mechanisms.The fi rst is the indirect one.It was discovered by Ducy and al. [3][4][5][6][7][8] in mutant mice and rats that either cannot produce or cannot respond to, leptin.Leptin secreted from fat cells is carried by the ObRa receptors of vascular endothelial cells across the blood-brain-barrier where it activates ObRb receptors in the hypothalamus.These signals then stimulace expression of HOBIF (hypothalamic osteoblast inhibitory factor) which when released, lowers the matrix-making ability of osteoblasts [2][3][4][5][6][7][8] .These authors discovered what they believed to be the reason why obese Ob(Lep) -/-mice, which should have low bone mass because of their lack of leptin, and thus estrogen, actually have an abnormally high bone mass [3][4][5][6][7][8] .They found that intracerebroventricular injections of leptin actually caused the bone mass to decrease.It is assumed that leptin stimulates the brain to release HOBIF.Another part of central control mechanism of leptin is the NPY and its Y2 receptor, which may stimulate HOBIF release.In mice lacking neuropeptide 2 (NPY2) receptors in the hypothalamus deletion of hypothalamic NPY2 receptors resulted in increased bone mass without any obvious endocrine imbalance that would aff ect bone homoeostasis, which shows that these receptors regulate bone formation by alterating autonomic function 9 .A loss of either Y2 receptors or leptin should reduce HOBIF and increase osteoblast productivity 9 .Elefteriou et al. 10 found that NPY-defi cient mice have normal bone mass, which is in contrast to the mice defi cient in NPY2 receptor.NPY4 receptor defi cient mice have normal bone mass, whereas double knockout of NPY2 and NPY4 receptors leads to a greater increase in bone volume than in mice defi cient only in the NPY2 receptor 11 .Both receptors modulate sympathetic outfl ow in the brain stem.Augmented bone formation in the double knockout mouse suggests synergistic activity between NPY2 and NPY4 receptor pathways 11 .The second direct mechanism of leptin exerts its osteotrophic eff ects by promoting diff erentiation of bone marrow stromal cells into osteoblasts 12 and by inhibiting osteoclast generation 13 .Circulating leptin penetrates the bone marrow, where it joins the autocrine/ paracrine leptin from early cells of the osteoblast lineage and from late-stage, matrix-mineralizing osteoblasts or from early osteocytes to stimulate the production of IGF-1.IGF-1 in turn stimulates the proliferation of osteoblast precursors makes the osteoblastic lineage cells more resistant to apoptosis, and further enhances bone formation through suppression of osteoclast generation by causing osteoblastic stromal cells to make less RANKL (NF-kap-paB ligand) which stimulates osteoclast generation, and by stimulating the production of the osteoclast-suppressing OPG (osteoprotegerine) the osteoclast suppressor.Osteoblasts freshly isolated from adult humans have functional ObRb receptors 14 .Bassilana et al. 15 have found that human mesenchymal stem cells begin expressing both the nonfunctional, leptin transvascular-transporting ObRa, the fully functional long ObRb signal-transducing receptors, and leptin itself during osteoblastic diff erentiation.Reseland te al. 16 have found that human osteoblasts start making and secreting leptin when they are either in the late, matrix-mineralizing stage or when changing to osteocytes.Leptin has also stimulates the proliferation of cultured human osteoblasts 17 , and it has been shown to cause human bone marrow stromal cells to express alkaline phosphatase, collagen-I, and osteocalcin and to mineralize matrix 12 .
Although data from cultured human bone cells point to leptin as a direct stimulator of bone growth and a suppressor of bone resorption, the correlation between the serum leptin level and BMD (bone mineral density) in humans is so far equivocal, possibly because of a shifting balance between the cytokine's stimulatory direct action and sup-pressive indirect action on bones via the hypothalamus.In human beings, positive, negative, or no associations between serum leptin levels and bone mineral density have been reported.Pasco et al. 18 have reported that serum leptin levels are positively correlated to the bone mineral content.Results of Blain et al. 19 demonstrated that leptin is an independent predictor of whole body and femoral neck BMD in postmenopausal women.Yamauchi et al. 20 showed that in postmenopausal women plasma leptin levels were positively correlated with BMD values.Moreover, plasma leptin levels were signifi cantly lower in women with vertebral fractures than in those without fractures.The study of Di Tomaselli et al. 21also confi rms a significant correlation between serum leptin and BMD in early postmenopausal women, but the correlation was lost during progression of the postmenopausal period.Thomas et al. 22 have reported that BMD is positively correlated to fat mass and the serum leptin level in women.Z. Nagy et al. 23 found a negative correlation between serum leptin levels and radial and femoral BMD in postmenopausal women.On the other hand P. Hadji et al. 24 reported that bone mass is not correlated with the serum leptin level in pre or postmenopausal women.Goulding et al. found no signifi cant correlations between plasma leptin and dynamic markers of bone resorption or formation 25 .Rauch et al. also failed to fi nd a relationship between bone mass and serum leptin levels by examining total and trabecular bone density at the distalradius in adult women and reported no correlation, between plasma leptin level and bone turnover markers in adult women 26 .

Diff erences in leptin levels between men and women
Leptin possesses dimorphism, it is higher in women than in men.Leptin pulse amplitude is two to three times higher in women than in men and the expression rate of leptin mRNA in subcutaneous fat tissue is signifi cantly higher in females than in males 27 .Moreover, in vitro, the leptin secretion rate of men's subcutaneous adipose tissue is 66 % that of women's 28 .This would suggest a dependence of leptin on estrogen.Some authors admitt the diff erence may result not from higher estrogen level in women, rather from a suppressive eff ect of high levels of circulating androgens in men on leptin production 29,30 .It has been suggested that androgens reduce the regulation of leptin secretion 29,31 .Administration of testosterone reduces the expression of leptin mRNA in men.However, the infl uence of circulating androgens is not supported by other studies 33,34 .In these studies, leptin levels were invertly correlated with androgen levels.

Leptin and menopause
Postmenopausal women, whose estrogen levels naturally or artifi cially decline, are at the highest risk for developing osteopenia and osteoporosis.The infl uence of hormonal changes during menopause on leptin levels is still poorly understood.It is well documented that the serum concentration of leptin secreted by adipocytes correlates positively with the amount of fat mass and BMI 35- 38 .It appears that percent body fat in postmenopausal Action of leptin on bone and its relationship to menopause women may be the best adiposity-related predictor of serum leptin 39 .As a consequence of the decline in endogenous estrogens and reduced energy during menopause women tend to gain body fat 40 .After menopause heavier women conserve bone mass better than those with lower body weight.The protective eff ect of obesity on bone mass has been ascribed to a high body fat content.As a result of menopause transition the levels of circulating estrogens decrease whereas the adrenal glands continue to secrete androgen precursors.Inactive steroid precursors are aromatized to estrogens in adipose tissue.The relative increase in the androgen/estrogen ratio is likely to be important for the fat distribution shift 41 .Postmenopausal women manifest shift to a abdominal fat distribution (android type of obesity), which is associated with a more androgenic profi le with increased free testosterone levels and lower sex hormone-binding globulin (SHBG) levels.The shift to abdominal adiposity shows itself in the changes of WHR (waist to hip ratio).The connection between serum leptin concentration and WHR is controversial.Some authors have shown that serum leptin levels are higher in android obese women than in gynoid ones 42 , but other studies demonstrate a lack of correlation between serum leptin concentration and the type of obesity.Martinez-Carpio et al. found no correlation between leptin and postmenopausal women, but they found signifi cant correlations between leptin and weight, BMI and chest, waist and hip circumferences 43 .This is in agreement with the results of Tupikowska and Minocci et al. 34,44 .They also showed that WHR values were not correlated with leptin concentrations 44 .They suggest that subcutaneous abdominal fat is a determinant of leptin concentration, independently of the amount of fat mass, whereas the contribution of preperitoneal visceral fat is not signifi cant.
Various studies have shown diff erences in leptin values in pre-and postmenopausal women.Some authors suggest that serum leptin concentration are lower in the post-menopausal period.Rosenbaum et al. 29 Shimizu et al. 45 They report that pre-menopausal women have higher leptin production than postmenopausal women.However there are also reports indicating that decrease in estrogen levels in postmenopausal status has no direct eff ect on adipose tissue leptin production or concentrations 31,34,[45][46][47][48] .There are only few studies comparing the leptin level in women with artifi cially induced menopause before and after ovarectomy.When women with previously normal cycles before surgery were studied during the fourth postoperative day period after bilateral ovariectomy a significant reduction in leptin concentrations was found.There was a positive correlation of leptin values with estradiol and progesterone before and after the operation 49 .
Several authors have investigated plasma leptin levels in postmenopausal women during administration of HRT (hormone replacement therapy).Some results show that estrogens can regulate leptin production and its serum concentration 45 .Some studies reported no diff erences in serum leptin levels between users and non -users of hormonal replacement therapy, while others showed both de-creased and increased leptin levels.Existing clinical data addressing this issue are discordant.Di Carlo et al. 50found increased serum leptin level in untreated postmenopausal women decreased to premenopausal values after hormonal treatment.These results suggested that sex hormone replacement therapy can modulate leptin levels.In obese and non-obese postmenopausal women studied before and after HRT administration, plasma leptin levels showed an increase following treatment which was more pronounced in obese women 51 .Also intranasal and oral HRT had the eff ect of increasing the levels of leptin after 24 weeks in healthy postmenopausal women 52 .Other authors indicate that neither endogenous E2 nor sex hormone replacement therapy has any eff ect on serum leptin concentration 34 .These results support the hypothesis that the expression of leptin in adipocytes and circulating levels of this hormone are not related to the estrogen levels and type of hormonal therapy used.Hickey et al. analyzed data on postmenopausal women taking part in a cardiovascular risk factor study and found that leptin concentrations were similar in sex hormone users and non-users 53 .Also the results of the San Antonio heart study also showed no relation between serum leptin levels and menopausal status or postmenopausal sex hormone use 54 .Gower et al. compared HRTtreated postmenopausal women with untreated controls.There were no signifi cant determinants of serum leptin levels 55 .In a randomized placebo-controlled study in postmenopausal women with type 2 diabetes mellitus treated with transdermal E2 +NETA (estradiol + norethisteorne acetate), no signifi cant alterations in leptin levels were found 56 .The group of Di Tomaselli evaluated serum leptin levels in healthy HRT treated and untreated postmenopausal women.In women taking HRT, serum leptin levels did not change signifi cantly throughout the study period 57 .In an large sample of healthy postmenopausal users and non-users of hormone replacement therapy, Hadji et al. showed that neither sex hormone supplementation nor serum estrogen concentrations had any eff ect on leptin concentration, even after adjustment for BMI 24 .Other investigators take the possibility into account that diff erences in leptin level may depend on the type and dose of HRT used 58 .Gower et al. measured leptin in women with an untreated spontaneous cycle and with a cycle treated withdiff erent types of HRT.Women treated with estradiol plus progesterone had elevated serum leptin concentrations, but when estradiol was administered alone, leptin concentrations were not aff ected 56 .Serum leptin levels were increased in postmenopausal women treated with short-term HRT independent of changes in fat mass; in the same women, progesterone administration alone did not infl uence leptin levels.Some authors suggest that only a supraphysiological estrogen concentration can act on adipocytes and modulate leptin production.It is possible that diff erent doses of estradiol exert a more pronounced eff ect on circulating leptin 59 .Also studies in women with artifi cial menopause show similar results.Messinis et al. studied leptin levels in ovariectomized normal women treated with estradiol plus progesterone 60 .They found that HRT prevented the ovariectomy -induced decrease in lep-M.Petzel tin levels 59,61 .On the other hand administration of HRT to ovariectomized women starting 15 days after surgery did not modify leptin levels 62 .

Leptin administration as a treatment for osteopenia
Leptin might be used to treat osteopenia only if it can be engineered to avoid suppressive indirect eff ects on bones via the hypothalamus -avoid the hypothalamus and refrain from stimulating HOBIF expression.Only a very small group of humans is knowm to possess homozygous mutations for the leptin gene.Up to date, two United Kingdom families of Pakistani origin carrying a frameshift/premature mutation, and one Turkish family carrying a missense mutation, have been described 21 .This mutation leads to a constant demand for food, resulting in severe obesity.This condition can be successfully treated by the administration of recombinant human leptin and theroretically administration of leptin could be used to treat osteopenia in these subjects.

CONCLUSION
Although data from cultured human bone cells point to leptin as a direct stimulator of bone growth and a suppressor of bone resorption, understanding of leptin's role in human bone-remodelling is not yet defi ned.The exact role of leptin on bone mass remains unclear.The correlation between the serum leptin levels and BMD in humans is so far equivocal, possibly because of a shifting balance between the cytokine's stimulatory direct action and suppressive indirect action on bones via the hypothalamus.The eff ect that oestrogen decrease and deficiency during menopause and administration of hormone replacement therapy has on leptin production and contribution of leptin on emergence of osteopenia remains controversial.Existing clinical data addressing this issue are discordant.Larger clinical studies are therefore necessary to clarify leptin's role in vivo and to assess the contribution of the central and peripheral role of leptin to the overall maintenance of bone turnover in human beings and how leptin contributes to the mechanism of loss of bone mass during artifi cial and natural menopause.