Leptin Inhibits Bone Formation through a Hypothalamic Relay

Cell, Vol. 100, 197–207, January 21, 2000, Copyright 2000 by Cell Press

Leptin Inhibits Bone Formation through a Hypothalamic Relay: A Central Control of Bone Mass Patricia Ducy,*k Michael Amling,†k Shu Takeda,* Matthias Priemel,† Arndt F. Schilling,† Frank T. Beil,† Jianhe Shen,* Charles Vinson,‡ Johannes M. Rueger,† and Gerard Karsenty*§ * Department of Molecular and Human Genetics Baylor College of Medicine Houston, Texas 77030 † Department of Trauma Surgery University of Hamburg Hamburg 20246 Germany ‡ Laboratory of Biochemistry National Cancer Institute Bethesda, Maryland 20892

Summary Gonadal failure induces bone loss while obesity prevents it. This raises the possibility that bone mass, body weight, and gonadal function are regulated by common pathways. To test this hypothesis, we studied leptin-deficient and leptin receptor–deficient mice that are obese and hypogonadic. Both mutant mice have an increased bone formation leading to high bone mass despite hypogonadism and hypercortisolism. This phenotype is dominant, independent of the presence of fat, and specific for the absence of leptin signaling. There is no leptin signaling in osteoblasts but intracerebroventricular infusion of leptin causes bone loss in leptin-deficient and wild-type mice. This study identifies leptin as a potent inhibitor of bone formation acting through the central nervous system and therefore describes the central nature of bone mass control and its disorders. Introduction Bone mass is regulated through bone remodeling. The fundamental importance of this process in vertebrate biology is illustrated by the fact that osteoporosis, a bone remodeling disease, is the most common disease in the Western Hemisphere (Cooper and Melton, 1996). Bone remodeling is comprised of two well-defined cellular events that occur sequentially. There is first resorption of preexisting bone by the osteoclasts then de novo bone formation by the osteoblasts (Frost, 1969). The two phases of bone remodeling occur in a balanced fashion in order to maintain bone mass within narrow limits between the end of puberty and arrest of gonadal function. The prevailing view is that bone remodeling is governed primarily by autocrine/paracrine mechanisms. However, several lines of evidence suggest the involvement of endocrine/systemic regulators in the control of § To whom correspondence should be addressed (e-mail: karsenty@

bcm.tmc.edu). k These authors contributed equally to this work.

bone remodeling. For instance, gonadal failure and the sex steroid deficiency accompanying it stimulate the bone resorption phase of bone remodeling (Riggs and Melton, 1986). Moreover, novel regulators of bone resorption acting in a systemic manner have been identified recently. Osteoprotegerin is a circulating protein inhibiting osteoclast differentiation whose functional characterization was first performed by raising its serum concentration (Simonet et al., 1997). Likewise, systemic injection of osteoprotegerin ligand, a stimulator of osteoclast differentiation, increases bone resorption (Lacey et al., 1998). In contrast to what has been learned about bone resorption, we still know very little about the genetic control of bone formation by the osteoblasts. The existence of a systemic or endocrine regulation of bone resorption suggests that a similar mechanism may control bone formation, although it has not been identified genetically. The identification of systemic regulators of bone formation, if they exist, is of paramount importance given the incidence and morbidity of low bone mass diseases and the current lack of anabolic therapies for these disorders. To identify regulators of bone formation, we isolated in a classical bone remodeling disease features allowing us to formulate a hypothesis testable genetically and physiologically. Osteoporosis, the most frequent bone remodeling disease, is defined by a low bone mass and a high risk of fractures (Consensus Development Conference, 1993). It is caused by a relative increase of bone resorption over bone formation. Two clinical characteristics of osteoporosis can be used to propose a hypothesis. First, osteoporosis invariably develops following gonadal failure (Riggs and Melton, 1986; Riggs et al., 1998). Second, obesity protects from osteoporosis through an unknown mechanism (Felson et al., 1993; Tremollieres et al., 1993; Ravn et al., 1999). In molecular terms, together these two observations suggest that bone mass, body weight, and gonadal function could be regulated by similar molecules. Leptin is a small polypeptide hormone secreted primarily by the adipocytes. It controls body weight and, directly or indirectly, gonadal function following its binding to a specific receptor located in the hypothalamus (see Spiegelman and Flier, 1996 and Friedman and Halaas, 1998 for review). Accordingly, ob/ob and db/db mice that are deficient in leptin or its receptor, respectively, are obese and hypogonadic (Zhang et al., 1994; Tartaglia et al., 1995). This hypogonadism should lead to a low bone mass phenotype as gonadal failure favors bone resorption over bone formation (Riggs and Melton, 1986). The hypercortisolism of these animals is another osteoporosis-favoring condition by inhibiting bone formation (Reid, 1997; Ahima et al., 1999). Instead, we show here that the absence of leptin signaling in ob/ob and db/db mice results in a high bone mass (HBM) phenotype whose appearance precedes the onset of obesity. ob/ob and db/db mice are identified

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Figure 1. High Bone Mass Phenotype in ob/ ob and db/db Mice (A) X-ray analysis of vertebrae (vert.) and long bones (femurs) of 6-month-old wt and ob/ob mice. (B) Histological analysis of bones of 3-monthold (3 m.) and 6-month-old (6 m.) wt and ob/ ob mice. Mineralized bone matrix is stained in black by the von Kossa reagent. Note the increased number of black trabeculae in ob/ ob mice compared to wt littermates. (C) Quantification of the increase in bone volume in ob/ob mice. BV/TV, bone volume over trabecular volume. Gray bars, wt mice; black bars, ob/ob mice. (D) Three-point bending analysis of femurs from wt, ob/ob, and wt ovariectomized (wtOVX) mice. The ob/ob mice have a higher percentage of load to failure than wt ovariectomized animals. (E) Histological analysis of vertebrae of 6-month-old wt and db/db mice. (F) Quantification of the increase in bone volume in db/db mice. (G) Comparison of the hormonal profiles of wt and ob/ob mice. ob/ob mice are hypercortisolic. (PTH, pg/ml; thyroxin (T4), ␮g/dl; IGF1, ng/ml; corticosterone, ng/ml.) (H) Bone remodeling in absence of leptin signaling. Despite the existence of hypogonadism and hypercortisolism, two bone loss– favoring conditions, ob/ob and db/db mice have a high bone mass. Asterisks indicate a statistically significant difference between two groups of mice (p ⬍ 0.05). Error bars represent SEM.

animal models in which hypogonadism and hypercortisolism coexist with HBM. Histomorphometric and physiologic analyses demonstrate that leptin is a selective inhibitor of bone formation. There is no evidence of leptin signaling in osteoblasts, fat is not required for the development of this phenotype, but intracerebroventricular infusion of leptin reverses completely the HBM phenotype of the ob/ob mice and induces bone loss in wildtype (wt) mice. These findings describe a central regulation of bone remodeling. This novel regulatory loop describes a mechanism able to overcome the deleterious effects of gonadal failure and hypercortisolism on bone mass. These results should modify our understanding of bone remodeling and of osteoporosis. Results High Bone Mass Phenotype Despite Hypogonadism and Hypercortisolism in ob/ob and db/db Mice Without any known exception, hypogonadism induces an increase in osteoclast number and in bone resorption activity; as a result, it leads to a low bone mass phenotype (Riggs and Melton, 1986). Thus, the ob/ob mice that have a hypogonadism of hypothalamic origin should

have a lower bone mass than wt littermates (Ahima et al., 1996; Chehab et al., 1996; Ahima et al., 1997). To determine if the obesity of the ob/ob mice could affect their expected low bone mass phenotype, we performed X rays of vertebrae and long bones of 6-month-old wt and ob/ob mice. Surprisingly, the bones of the ob/ob mice appeared much denser than those of their wt littermates (Figure 1A), demonstrating the presence of a higher amount of mineralized bone matrix. Given the poor sensitivity of X rays to quantitate bone mass abnormalities, this increased bone density was an indication of a major change in bone architecture. Histologic analyses performed in 3- and 6-month-old mice showed the presence of many more thick trabeculae in the bones of ob/ob mice compared to those of wt mice (Figure 1B). The cortical bone was not affected (data not shown). Histomorphometric quantitation showed a nearly 2-fold increase in trabecular bone volume in ob/ob mice compared to wt littermates (Figure 1C). This phenotype was observed in both sexes. The functional consequences of this increase in bone mass were analyzed by comparing the biomechanical properties of long bones of 6-month-old ob/ob mice, wt mice, and wt mice that were ovariectomized (wt-ovx) for 4 months

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Figure 2. The High Bone Mass Phenotype of the ob/ob Mice Is Due to Leptin Deficiency, Not to Obesity (A) Histological analysis of vertebrae of 1-month-old wt and ob/ob mice fed a low fat diet (LF diet). The body weight (B. Weight) is similar between both groups of mice, but the bone volume (Bone Vol., BV/TV) is increased in ob/ob mice. (B) Histological analysis of vertebrae of 3-month-old wt and ob/⫹ mice. While there is no difference in body weight, heterozygote mutant mice have an increase in bone volume. (C) Histological analysis of vertebrae of 6-month-old wt and Agouti yellow mutant mice (Ay/a). The body weight is higher in mutant mice, but there is no difference in bone volume compared to wt mice. (D) Histological analysis of vertebrae of 6-month-old wt mice fed a normal diet or a high fat (HF) diet. Bone volume was not increased in mice fed HF diet despite a 50% increase in their body weight. Underlined numbers indicate a statistically significant difference between experimental and control groups of mice (p ⬍ 0.05).

to mimic the hypogonadic status of the ob/ob mice. Using a three-point bending assay, failure load of the bones from wt and ob/ob mice were indistinguishable but significantly higher than the one observed in the bones of wt-ovx mice (Figure 1D), indicating that leptin deficiency has a beneficial effect on the biomechanical properties of the bones. We also analyzed the bones of the db/db mice that have an inactivating mutation of the leptin receptor (Tartaglia et al., 1995). Like the ob/ob mice, the db/db mice are obese and hypogonadic. There was in db/db mice an increase in the number of trabeculae in both long bones and vertebrae similar to that observed in ob/ob mice (Figure 1E and data not shown), resulting in a 3-fold increase in bone volume compared to wt mice (Figure 1F). This latter result demonstrates that leptin signals through its known receptor to affect bone mass. To determine whether the HBM phenotype of the ob/ ob mice could be explained by other endocrine abnormalities secondary to the absence of leptin signaling, we measured in their serum the level of hormones whose dysregulation affects bone mass (Figure 1G). ob/ob mice had a normal level of parathyroid hormone, a hormone whose deficiency causes HBM. They also have nearly normal IGF1 and thyroxin levels but a severe hypercortisolism (Figure 1G and Ahima et al., 1999). This is of

critical importance since hypercortisolism inhibits bone formation and is the second most frequent cause of bone loss after gonadal failure. Thus, the HBM phenotype of the ob/ob mice develops despite the coexistence of two bone loss–favoring circumstances, hypogonadism and hypercortisolism (Figure 1H). The uniqueness of this situation led us to explore its underlying mechanisms.

The High Bone Mass Phenotype of the ob/ob Mice Is Not Secondary to Obesity The HBM phenotype of the ob/ob and db/db mice could be secondary either to a lack of leptin signaling or to the obesity of these mice. To distinguish between these two possibilities, we analyzed several additional groups of mutant mice. First, we took advantage of the fact that a low fat diet postpones the appearance of obesity in ob/ob mice. ob/ob mice fed this low fat diet had a normal weight at one month of age; nevertheless, they already had an HBM phenotype at that age (Figure 2A). Second, we analyzed heterozygote leptin–deficient mice (ob/⫹) that are not obese; these animals also had an HBM phenotype (Figure 2B). Third, we studied the bones of other mouse models of obesity that are not primarily related to leptin signaling. Another genetic model of

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Figure 3. The Absence of Leptin Signaling Causes an Increase in Osteoblast Function (A) Calcein double labeling in 3-month-old wt and ob/ob mice. The distance between the two labels (white arrow) represents the rate of bone formation. It is significantly increased in ob/ob mice. (B–F) The rate of bone formation is increased in ob/ob mice (B) and db/db mice (D) 45% and 70%, respectively, compared to wt littermates. This increase occurs in the presence of a normal number of osteoblasts (C and E) and in spite of the increased number of osteoclasts due to their hypogonadism (F). Empty bars, wt mice; black bars, ob/ob mice; gray bars, db/db mice; 3 m., 3-month-old animals; 6 m., 6-month-old animals. (G) Increased bone formation rate in fatrestricted 1-month-old ob/ob mice and heterozygote ob/⫹ mice, which are not obese (see body weights Figures 2A and 2B). (H) wt mice fed a high fat diet, or Ay/a mice that are overweight (see Figures 2C and 2D) but not leptin deficient have a normal rate of bone formation. Asterisks indicate statistically significant differences compared to control mice (p ⬍ 0.05). Error bars represent SEM.

obesity, the Agouti yellow (Ay/a) mice (Herberg and Coleman, 1977), had a normal bone mass; the same was true for wt mice fed with a high fat diet (Figures 2C and 2D). In summary, the existence of an HBM phenotype in ob/ob mice prior to the appearance of obesity and in ob/⫹ mice that are not obese as well as its absence in leptin-unrelated models of obesity demonstrate that it is the absence of leptin signaling, not the obesity, that causes this HBM phenotype. Increased Osteoblastic Function in ob/ob and db/db Mice The increase in bone mass in these mice could be due to an increase in osteoblastic bone formation, to a decrease in osteoclastic bone resorption, or to a combination of both abnormalities. To study osteoblast function in vivo, the bone formation rate was quantified following double labeling with calcein, a marker of newly formed bone (Figure 3A). In 3- and 6-month-old ob/ob mice, there was a 70% and 60% increase, respectively, of the bone formation rate compared to that of wt littermates (Figure 3B). This result demonstrated that the HBM phenotype of the ob/ob mice was due, at least in part, to an increase in bone formation activity. Remarkably, considering the massive increase of the bone formation rate, the osteoblasts surface as well as the osteoblast number were not increased in ob/ob mice, indicating that leptin deficiency affects the function of the osteoblasts, not their differentiation or proliferation after birth (Figure 3C). The same observations were made in db/ db mice (Figures 3D and 3E). As expected given the existence of a hypogonadism, the number of osteoclasts was increased in both mutant mouse strains (Figure 3F), suggesting that their HBM phenotype has developed despite an increase in bone resorption. We ascertained the specificity of the effect of the

absence of leptin signaling on osteoblast function using the same control animals as above. One-month-old ob/ ob animals that were fed a low fat diet and heterozygote leptin–deficient mice, both lean, had a significant increase in their rate of bone formation (Figure 3G). In contrast, Ay/a mutant mice as well as wt mice fed a high fat diet, two leptin-unrelated models of obesity, had normal bone formation parameters (Figure 3H). Analysis of Osteoclast Function in ob/ob Mice The coexistence of an HBM phenotype and of hypogonadism was so exceptional that it raised the hypothesis that bone resorption might be defective in these mice. The increased urinary elimination of deoxypyridinoline crosslinks (Dpd), a biochemical marker of bone resorption (Eyre et al., 1988), in the ob/ob mice argued against this hypothesis (wt: 10.5 ⫾ 2.5 nM Dpd/mM creatinine; ob/ob: 24.0 ⫾ 4.0 nM Dpd/mM creatinine). To address this point more thoroughly, we took advantage of the hypogonadism of the ob/ob mice. We reasoned that if the osteoclasts of the ob/ob mice were functional, correcting their hypogonadism should decrease their rate of bone resorption by diminishing the number of osteoclasts and thereby further increasing their bone mass. On the other hand, if the osteoclasts of the ob/ ob mice were not functioning properly, correcting their hypogonadism should not affect the severity of their HBM phenotype. To determine which of these two possibilities was correct, estradiol or placebo pellets were implanted subcutaneously in 2-month-old female ob/ ob mice; these animals were analyzed after a 3-month treatment period. As expected, the estradiol treatment corrected the hypogonadism of these mice as judged by the aspect of their uteri and their levels of estradiol in blood (Figure 4A and data not shown). It also led to a normalization of the osteoclast number and to a further increase of

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an assay using hematopoietic progenitor cells from wt, ob/ob, or db/db mice and growth factors known to induce the differentiation of these cells into functional osteoclasts. As shown in Figure 4E, wt, ob/ob, and db/ db hematopoietic progenitor cells differentiated equally well into osteoclasts able to resorb a matrix. These experiments demonstrate that there is no detectable functional defect of the osteoclasts in ob/ob and db/db mice and establish that the HBM phenotype of these mouse mutant strains results exclusively from the increase in bone formation secondary to the absence of leptin signaling.

Figure 4. Normal Osteoclasts Function in Absence of Leptin Signaling (A–D) Comparative analyses of wt and ob/ob mice whose hypogonadism has been corrected by 17␤-estradiol treatment (E2) or not corrected (P, placebo). (A) Correction of the uterus atrophy of the ob/ob mice by the 17␤estradiol treatment. (B–D) Histological analysis of vertebrae showing that 17␤-estradiol treatment leads to a significant increase in bone trabeculae in 17␤estradiol–treated wt and even more in 17␤-estradiol–treated ob/ob mice (B and D). The increased bone volume is more pronounced in 17␤-estradiol–treated ob/ob mice than in treated wt mice compared with placebo-treated animals (D), as their number of osteoclasts is returned to a normal range (C). Gray bars, wt mice; black bars, ob/ ob mice; patterned bars, treated mice; solid bars, placebo control mice. Asterisks indicate a statistically significant difference between treated and untreated mice (p ⬍ 0.05). Error bars represent SEM. (E) Normal differentiation and function of ob/ob and db/db osteoclasts ex vivo. Marrow progenitors derived from wt, ob/ob, and db/ db mice differentiate equally well in TRAP positive (TRAP⫹) osteoclasts (upper panel and bottom line). There is also no difference in their ability to form resorption pits on a dentin slice matrix (bottom panel).

the HBM phenotype of these mice compared to placebo treated ob/ob mice (Figures 4B–4D). Similar results were obtained in male ob/ob mice treated with testosterone implants (data not shown). This finding excluded a defect of bone resorption as the origin of the HBM phenotype in ob/ob mice. Finally, the function of the osteoclasts of ob/ob and db/db mice was studied in vitro in

Absence of Leptin Signaling in Osteoblasts Our analysis indicates that leptin is an inhibitor of osteoblastic bone formation. Moreover, the existence of an HBM phenotype in db/db mice demonstrates that leptin must bind to its known receptor to fulfill this function. In theory, leptin could either act directly on osteoblasts, indirectly through the release of a second factor present in fat, or by using an hypothalamic relay as it does for the control of body weight. We tested these three possible mechanisms of action. We could not detect any expression of Leptin in osteoblasts even after a long exposure, indicating that an autocrine regulation was unlikely (Figure 5A). We also could not detect any Leptin expression in whole bone samples, providing an indirect argument against a paracrine regulation of osteoblast function by leptin (Figure 5A). In any case, a paracrine and/or endocrine regulation of osteoblast function by leptin would require that functional leptin receptors are present on osteoblasts. There are several transcripts of the leptin receptor but only one, Ob-Rb, is thought to have signal transduction ability (Tartaglia et al., 1995; Chen et al., 1996; Lee et al., 1996). The expression of this transcript of leptin receptor is highly, although not strictly, hypothalamus specific. RT-PCR experiments were performed to search for ObRb transcripts in primary osteoblasts and whole bone samples. In several experiments using a number of amplification cycles necessary to detect Ob-Rb transcripts in hypothalamus, we failed to detect Ob-Rb expression in calvaria, long bone, and primary osteoblast cultures (Figure 5B). To determine whether or not leptin could transduce its signal in osteoblasts, we treated serum-starved primary osteoblasts isolated from wt mice with leptin. We monitored the phosphorylation of Stat3, a downstream effector of leptin signaling in its target cells (Tartaglia et al., 1995; Baumann et al., 1996; Ghilardi et al., 1996; Vaisse et al., 1996), and the expression of two immediate early genes whose transcription increases following leptin treatment of target cells (Elmquist et al., 1997). As a positive control, we used oncostatin-M that also induces Stat3 phosphorylation and activates the expression of the same immediate early genes in osteoblasts (Levy et al., 1996). As shown in Figure 5C, treatment of osteoblasts with Oncostatin-M induced Stat3 phosphorylation while leptin treatment did not. Several doses of leptin, physiologic and supraphysiologic, were used in this experiment; all of them failed to induce Stat3 phosphorylation (data not shown). Similarly, expression of Tis11 and c-fos was quickly and transiently activated

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Figure 5. Leptin Does Not Signal in Osteoblasts (A) Northern blot analysis of Leptin expression in tissues and primary cells (nonmineralizing (NM) osteoblasts, mineralizing (M) osteoblasts, and chondrocytes) (upper panel). Leptin expression can be detected only in fat tissues, even after overexposure. Gapdh expression was used as an internal control for loading (lower panel). (B) Ob-Rb transcripts cannot be detected in long bones, calvaria, and primary osteoblasts by RT-PCR while this message is detected in hypothalamus. Amplification of Hprt was used as an internal control for cDNA quality and PCR efficiency. (C) Western blot analysis. Absence of Stat3 phosphorylation (phospho Stat3) upon treatment of serum-starved primary osteoblast cultures with leptin. Induction by oncostatin-M (OSM) was used as a positive control. Immunodetection of Stat3 shows that identical amounts of proteins were analyzed. (D) Northern blot analysis of immediate-early gene expression (Tis11 and c-fos genes) upon treatment of primary osteoblast cultures with leptin or OSM. An immediate and transient signal can only be observed in OSMtreated cultures. Gapdh expression was used as an internal control for loading (lower panel). min., minutes of treatment. (E) Ex vivo primary osteoblast cultures from wt mice maintained in the absence (vehicle) or presence of leptin. No effect on collagen synthesis (upper panel, van Gieson staining) or matrix mineralization (lower panel, von Kossa staining) can be observed. (F) Normal function of db/db osteoblasts in ex vivo culture experiments. No difference can be observed on collagen synthesis (upper panel, van Gieson staining) or matrix mineralization (lower panel, von Kossa staining) between primary osteoblast cultures derived from wt and db/ db mice.

by oncostatin-M but not by leptin (Figure 5D). Lastly, we studied the effect of a long-term leptin treatment of primary osteoblast cultures from wt mice on extracellular matrix synthesis and bone matrix mineralization. No difference was observed when assessing collagen synthesis or formation of mineralization nodules between control and leptin-treated cultures (Figure 5E). If there is no functional leptin receptor on osteoblasts, then wt and db/db osteoblasts should be undistinguishable in ex vivo culture. Indeed, for all the parameter analysis, alkaline phosphatase staining, type I collagen production, and formation of mineralization nodules, there was no difference between wt and db/db primary osteoblast cultures (Figure 5F and data not shown). Taken together, these results indicate that leptin action on bone formation in the entire animal does not require leptin binding to a receptor located on the osteoblasts. High Bone Mass in Absence of Fat To address the possibility that this action of leptin could require the presence of fat, we made use of a transgenic mouse model expressing, exclusively in adipocytes, a dominant-negative protein termed A-ZIP (Moitra et al., 1998). This dominant-negative protein abolishes DNA binding of most B-ZIP transcription factors, a class of transcription factors critical for adipocyte differentiation. As a result, the A-ZIP/F-1 transgenic mice have no

white adipose tissue, which is the type of fat regulated by leptin signaling, and they have dramatically reduced amounts of inactive brown adipose tissue. They also have a 20-fold reduction in leptin level (Moitra et al., 1998). Yet, A-ZIP/F-1 transgenic mice have the same HBM phenotype due to an increase in osteoblast function found in ob/ob and db/db mice (Figure 6). This experiment has two implications. First, it confirms that leptin deficiency, not high fat index, is responsible for the HBM phenotype of the ob/ob and db/db mice. Second, it demonstrates that fat is not a necessary relay for the action of leptin on bone formation. Bone Loss Following Intracerebroventricular Infusion of Leptin in ob/ob and wt Mice We next addressed whether leptin binding to its hypothalamic receptor could correct the HBM phenotype of the ob/ob mice as it can rescue their obesity phenotype. We inserted pumps delivering either PBS or leptin (8 ng/hr) into the third ventricle of ob/ob mice. This dose has been shown to have no effect when administered systemically (Halaas et al., 1997). To be as close as possible of the biologic situation of the ob/ob mice, the animals in which pumps were inserted were ovariectomized to avoid any increase in bone mass due to the correction of their hypogonadism. The pumps were left in place for 28 days. Classical histology showed that

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Figure 6. Fat Tissue Is Not Required for the Appearance of a High Bone Mass Phenotype (A) Histological analysis of vertebrae of 6-month-old wt and A-ZIP/ F-1 transgenic mice that have no fat tissue. Both bone volume (B) and bone formation rate (C) are increased in the transgenic mice. Asterisks indicate a statistically significant difference between wt and transgenic mice (p ⬍ 0.05). Error bars represent SEM.

the bone of leptin-treated mice but not of the PBStreated mice had regained a normal appearance (Figure 7A). Indeed, bone volume was significantly decreased in leptin-treated mice (Figure 7A). We could not detect any leptin in the serum of these animals using a specific radioimmunoassay (data not shown). To determine if leptin could affect bone mass in a physiologic situation, we performed the same experiment in wt mice. Classical histology showed bone loss in leptin-treated but not in PBS-treated wt mice and histomorphometric analyses showed that the bone volume was significantly decreased in leptin-treated mice (Figure 7B). In summary, the effect of leptin intracerebroventricular (icv) infusion on the bone mass of ob/ob and wt mice demonstrates that control of bone formation is a central, leptin-dependent, function. Leptin and Neuropeptide Y Do Not Have Antagonistic Functions in the Control of Bone Formation The results presented above indicate that leptin is a powerful inhibitor of bone formation that acts centrally. One of the very few neuromediators for which there is physiologic and genetic evidence that it affects leptin’s action on body weight is NPY (see Elmquist et al., 1999 and Inui, 1999 for review). Indeed, NPY neurons express leptin receptors, NPY expression is increased in ob/ob mice but blunted upon leptin administration, NPY icv infusion in wt animals causes body weight gain, and NPY deficiency partially corrects the obesity phenotype of the ob/ob mice (Erickson et al., 1996). Thus, we reasoned that if NPY was also in the leptin-dependent pathway controlling bone formation, icv infusion of NPY in

Figure 7. Leptin and NPY icv Infusions Affect Bone Mass Histological comparison of vertebrae of 4-month-old ob/ob (A) and wt (B) mice infused centrally (third ventricle) with PBS or leptin. In both cases, leptin icv infusion induces a decrease in bone mass and bone volume. (C) Histological comparison of vertebrae of 4-month-old wt mice infused centrally with PBS or NPY. NPY icv infusion causes a decrease in bone mass and bone volume. Underlined numbers indicate a statistically significant difference between experimental and control groups of mice (p ⬍ 0.05).

wt mice should lead to an HBM phenotype. To our surprise, icv infusion of NPY caused bone loss not bone gain (Figure 7C), indicating that leptin and NPY do not antagonize each other’s function in the control of bone formation as they appear to do in body weight control. This result suggests that leptin may use different sets of mediators to control body weight and bone mass. Discussion This study demonstrates that leptin is a major regulator of bone formation. It provides in vivo evidence for a central regulation of bone remodeling, thus establishing a physiologic paradigm for a hormone affecting bone mass. This paradigm should influence our understanding of osteoporosis, as it indicates that it is, at least partly, a central disorder. Our findings also identify leptin as a molecular determinant whose absence can overcome the low bone mass phenotype caused by gonadal

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failure and hypercortisolism. Lastly, our study uncovers an unexpected discrepancy between the mechanisms of action of leptin in the control of body weight and of bone formation. Leptin Is a Regulator of Bone Formation A myriad of abnormalities has been described in ob/ob and db/db mice besides obesity. These include, among others, insulin resistance, diabetes, abnormal behavior, decreased immune function, and hypothermia (Ingalls et al., 1950). Leptin plays a major and specific role in each of these physiologic processes. Likewise, multiple lines of evidence establish that the HBM phenotype of the ob/ob and db/db mice is a direct consequence of the absence of leptin signaling, not of obesity. Indeed, this phenotype precedes the appearance of obesity in ob/ob mice and is not observed in other models of obesity, while heterozygote leptin-deficient mice that are not obese and A-ZIP/F-1 transgenic mice, which have no white fat and a very low level of circulating leptin, both have an HBM phenotype. This phenotype is due to an increase in bone matrix deposition by the osteoblasts, not to an increase of their number. Among the multiple endocrine abnormalities of ob/ob mice, one of them is hypercortisolism, a condition known to induce bone loss by decreasing bone formation (Reid, 1997). Yet, despite this hypercortisolism and their hypogonadism that should favor bone resorption, the ob/ob mice have an HBM phenotype. The fact that this bone phenotype occurs in such unfavorable circumstances and is present in heterozygous leptin–deficient animals indicate that leptin’s action on bone formation is not less important physiologically than its role in body weight control. Bone Remodeling as a Central Function Leptin is a hormone secreted by adipocytes and controls body weight through a hypothalamic relay (see Spiegelman and Flier, 1996 and Friedman and Halaas, 1998 for review). Several molecular, genetic, and physiologic lines of evidence indicate that it uses a similar hypothalamic relay to control bone formation by the osteoblasts. First, the ob/ob and db/db mice both have the same bone phenotype, indicating that leptin uses a similar receptor to control body weight and bone mass. The absence of Leptin expression in osteoblasts rules out an autocrine mechanism. Although Ob-Rb transcripts have been detected in immortalized osteoblastic cell lines using a high number of amplification cycles (Thomas et al., 1999), we were unable to detect this transcript in primary osteoblasts. The absence of ObRb transcripts in osteoblasts, the lack of evidence of leptin signaling in osteoblasts together with the normal behavior ex vivo of osteoblasts isolated from db/db mice indicate that the osteoblast is not the direct target of leptin action on bone formation. The HBM phenotype of the A-ZIP/F-1 transgenic mice demonstrates that fat tissue is not necessary for the regulation of bone formation by leptin. In contrast, continuous delivery of a small dose of leptin in the third ventricle completely rescues the bone phenotype of the ob/ob mice and leads to bone loss in wt mice. This latter result demonstrates that leptin binding to its hypothalamic receptor is sufficient to

affect osteoblast function and reveals the central nature of bone remodeling regulation. Beyond its hypothalamic relay, what is the mode of action of leptin? The low bone mass phenotype of patients with panhypopituitarism (Kaufman et al., 1992) together with the presence of multiple endocrine abnormalities that should favor bone loss in ob/ob and db/ db mice argue against a classical pituitary relay for leptin action on bone formation. The same appears to be true for leptin’s effect on body weight (Aschner, 1912; Elmquist et al., 1999). In that case, leptin action could be mediated through the autonomic nervous system (Inoue and Bray, 1977; Elmquist et al., 1999). Since multiple lines of evidence indicate that NPY is partly involved in counteracting leptin’s effect on body weight, we tested whether it could affect bone mass. Unexpectedly, icv infusion of NPY led to a low bone mass phenotype, as does leptin icv infusion in wt mice. This result is of critical importance because it suggests that leptin uses different pathways to control body weight and bone mass. Hierarchy of Pathways Controlling Bone Remodeling Bone remodeling is classically thought to be regulated by cell–cell interactions and cytokines acting in an autocrine or paracrine fashion. In light of this study, it appears that bone mass is also controlled by a central pathway. This central regulatory pathway is of critical importance since ob/ob and db/db mice have an HBM in spite of hypercortisolism and hypogonadism. No other known pathway can overcome these two osteoporosisfavoring conditions. This genetic observation should lead us to propose new concepts to study bone remodeling and its disorders. By analogy with the emerging knowledge regarding the control of bone resorption by several secreted molecules (Suda et al., 1999), it is likely that leptin is only one of many circulating molecules affecting bone formation. How are these novel regulatory pathways and the classical autocrine/paracrine regulation contributing to bone remodeling? Conceivably, leptin and other secreted molecules could regulate bone mass by affecting the synthesis or the function of local factors present in the bone microenvironment. Alternatively, these two pathways may act independently of each other. The distinction between these two possibilities will have to await the identification of the leptin downstream effectors. Obesity as a Protective Factor Against Osteoporosis How does our study relate to the observation that obesity protects from bone loss in humans? This may appear in apparent contradiction with the fact that Leptin expression and serum leptin levels are elevated in obese humans compared to lean individuals (Maffei et al., 1995; Considine et al., 1996). However, as is the case in type II diabetes patients who show insulin resistance (Moller and Flier, 1991), it appears that obese individuals are resistant to the biologic effects of leptin (Caro et al., 1996; Bjorbaek et al., 1998). This resistance may be due to a partial failure to transport leptin to the central nervous system in obesity. Thus, given the state of leptin

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resistance of obese patients, the observation that the absence of leptin signaling in mice leads to HBM provides a molecular hypothesis to explain the protective effect of obesity on bone mass in humans. Our observation provides the first evidence for a mechanism linking the control of bone mass with the regulation of body weight and possibly gonadal function. Besides the multiple physiologic questions raised by this study, the data suggest that pharmacological manipulation of the leptin pathway may be a novel therapeutic approach to prevent and/or treat osteoporosis by stimulating bone formation. Experimental Procedures Animals Breeders and mutant mice (C57BL/6J Lepob, C57BL/6J Leprdb, C57BL/6J Ay/a) were purchased from the Jackson Laboratory. Generation of A-ZIP/F-1 transgenic mice has been previously reported. Genotyping was performed according to established protocols (Chua et al., 1997; Moitra et al., 1998; Namae et al., 1998). Animals were fed a regular diet (Purina #5001) or, when indicated, a high fat/high carbohydrate diet (Bio-serv #F3282). Histologic and Histomorphometric Analyses Contact X rays were performed using a Faxitron (Phillips). Bone specimens were processed as described. Histological analyses were performed on undecalcified sections stained with the von Kossa reagent and counterstained with Kernechtrot (Amling et al., 1999). Double-labeling technique has been described (Amling et al., 1999): calcein was injected twice at 8-day intervals, and animals were killed 2 days later. Static and dynamic histomorphometric analyses were performed according to standard protocols (Parfitt et al., 1987) using the Osteomeasure Analysis System (Osteometrics, Atlanta). Statistical differences between groups (n ⫽ 4 to 6) were assessed by Student’s test. Biomechanical Analysis Femora were tested to failure by three-point bending on a servohydraulic testing machine (Zwick GmbHandCo) at a constant displacement rate of 10 mm/min (Amling et al., 1999). Failure load is a measure of the strength of the bones. Steroid Treatments Placebo or 17␤-estradiol time-released pellets (Innovative Research of America) were implanted subcutaneously in 6-week-old wt and ob/ob females to maintain a serum concentration of 250 pg/ml. Animals were sacrificed after 3 months. Cell Cultures Mouse osteoclasts were generated in vitro according to protocols previously reported (Simonet et al., 1997; Quinn et al., 1998). Bone marrow cells of wt, ob/ob, db/db mice were cultured at an initial density of 106 cells/well on 24-well tissue culture plates or dentin chips in ␣-MEM (Sigma) containing 10% FBS (Hiclone), murine M-CSF 40 ng/ml (Sigma), RANKL/ODF 25 ng/ml (Peprotech), 10⫺8 M dexamethasone (Sigma), and 10⫺8 1,25 dihydroxy vitamin D3. Medium was changed every other day. After 6 days of culture, cells were fixed in 3.7% formalin. Osteoclasts formed on plastic plates were stained for tartrate resistant acid phosphatase. Cells on dentin chips were removed by treatment with sodium hypochloride solution. Dentin chips were then stained with toluidine blue to visualize resorption pits. Primary osteoblast cultures from calvaria of newborn wt or db/db mice were established as previously described (Ducy et al., 1999) and maintained in mineralization medium (aMEM/0.1 mg/ml ascorbic acid/5 mM ␤-glycerophosphate) supplemented with 10% FBS. Cultures of mutant cells were maintained in this medium for 15 days before analysis. Cultures derived from wt mice were maintained for 10 days in this medium, then the percentage of serum was reduced to 0.5%, and the medium supplemented with 1.2 ␮g/

ml leptin (Sigma) or vehicle for 5 days. The presence of a collagenrich extracellular matrix and of mineralization nodules was assessed by whole-mount staining of the cultures by the van Gieson and von Kossa reagent (Ducy et al., 1999), respectively. Leptin Signaling Assays RT-PCR analysis of Ob-Rb expression was performed on randomprimed cDNAs using previously described specific primers (Friedman and Halaas, 1998) for 27 cycles. Subconfluent primary osteoblast cultures from wt mice were maintained for 4 days in 10% FBS mineralization medium then switched to 0.5% for 2 days and replaced daily. Medium was replaced 2 hr before a 20 min treatment with 80 ng/ml leptin (Sigma) or 40 ng/ml Oncostatin M (R&D Diagnostics) or vehicle. RNA extractions were performed using Triazol (GIBCO). Northern blots were performed with 15 ␮g of total RNA according to standard procedures. For Western blot analysis, cells were lysed, protein extracts were separated on a 7.5% SDS-PAGE and blotted on nitrocellulose (Bio-Rad) for immunoblotting assay. Analysis of Stat3 phosphorylation was performed using the PhosphoPlus Stat3 (Tyr705) Antibody kit (New England Biolabs) according to the manufacturer’s instructions. Protein and Hormones Measurements Deoxypyridinoline cross-links were measured in morning urines using the Pyrilinks-D immunoassay kit (Metra Biosystem). Creatinine values were used for standardization between samples (Creatinine kit, Metra Biosystem). Serum levels of hormones were quantified by radioimmunoassays using kits from Diagnostic system laboratories (17␤-estradiol and IGF1), Linco (leptin), Nichols Institute Diagnostics (PTH), and ICN Biomedicals (T4 and corticosterone). Intracerebroventricular Infusion Animals were anesthetized with avertin and placed on a stereotaxic instrument (Stoelting). The calvaria was exposed and a 0.7 mm hole was drilled upon bregma. A 28-gauge cannula (Brain infusion kit II, Alza) was implanted into the third ventricle according to the following coordinates: midline, ⫺0.3 AP, 3 mm ventral (0 point bregma). The cannula was secured to the skull with cyanoacrylate, and attached with Tygon tubing to an osmotic pump (Alza) placed in the dorsal subcutaneous space of the animal. The rate of delivery was 0.25 ␮l/hr (8 ng/hr) of leptin (Sigma) or 0.5 ␮l/hr (75 ng/hr) of NPY (Sigma) or PBS for 28 days. Acknowledgments The authors thank A. Beaudet, A. Bradley, and F. Ramirez for reading of the manuscript, M. Goldfarb for probes, P. Catala-Lehnen, D. Briem, and H. Ritzel for their contribution to the in vitro and histomorphometry analyses, and T. Holzmann for his assistance in performing the biomechanical tests. P. D. and G. K. are indebted to D. Shine for his advice. M. A. thanks G. Delling for his early support. This work was supported by NIH (AR45548 and DE11290) and March of Dimes grants (to G. K.) and a grant from the German Research Community to (J. M. R.). P. D. was supported by an Investigator Award from the Arthritis Foundation. A. F. S. and F. T. B. are fellows of the German Research Community. Received September 24, 1999; revised December 17, 1999. References Ahima, R.S., Prabakaran, D., Mantzoros, C., Qu, D., Lowell, B., Maratos-Flier, E., and Flier, J.S. (1996). Role of leptin in the neuroendocrine response to fasting. Nature 382, 250–252. Ahima, R.S., Dushay, J., Flier, S.N., Prabakaran, D., and Flier, J.S. (1997). Leptin accelerates the onset of puberty in normal female mice. J. Clin. Invest. 99, 391–395. Ahima, R.S., Bjorbek, C., Osei, S., and Flier, J.S. (1999). Regulation of neuronal and glial proteins by leptin: implications for brain development. Endocrinology 140, 2755–2762. Amling, M., Priemel, M., Holzmann, T., Chapin, K., Rueger, J.M., Baron, R., and Demay, M.B. (1999). Rescue of the skeletal phenotype of vitamin D receptor ablated mice in the setting of normal mineral

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