Leptin predicts bone and fat mass after accounting for the effects of diet and glucocorticoid treatment in piglets

The role of leptin in neonatal growth and bone metabolism has been investigated, but not simultaneously. The objectives of this study were to determine if leptin relates to bone mass during rapid growth; if consumption of maternal milk is related to elevated circulating concentrations of leptin resulting in higher fat mass; and if glucocorticoids result in higher fat mass and reduced bone mass due to elevated leptin. Thirty-two piglets were randomized to either a suckling or milk substitute plus either dexamethasone (DEX) or placebo injection for 15 days beginning at 5 days of age. Milk and blood samples were obtained at baseline, and after 15 days, blood was sampled again for measurement of leptin and bone biochemistry. Weight at baseline plus weight and length after 15 days were recorded, followed by measurement of whole body bone mineral content, bone area, and fat mass using dual energy x-ray absorptiometry. At baseline, plasma leptin was elevated in suckled piglets. Piglets that suckled had elevated fat mass as did those who received DEX. However, DEX resulted in suppressed weight and length, bone mass, and bone metabolism. Leptin was similar among groups after the 15 days. After accounting for body size and treatment effects, piglet plasma leptin was predictive of bone and fat mass. Leptin circulating early postnatally is linked to body composition, specifically fat and bone mass. Elevations in fat mass and reductions in bone mass observed after 15 days of DEX treatment are not related to leptin metabolism. Both human and porcine neonates share similar characteristics with respect to relationships of leptin with fat and bone mass.     

Central control of bone remodeling by neuromedin U

Bone remodeling, the function affected in osteoporosis, the most common of bone diseases, comprises two phases: bone formation by matrix-producing osteoblasts and bone resorption by osteoclasts. The demonstration that the anorexigenic hormone leptin inhibits bone formation through a hypothalamic relay suggests that other molecules that affect energy metabolism in the hypothalamus could also modulate bone mass. Neuromedin U (NMU) is an anorexigenic neuropeptide that acts independently of leptin through poorly defined mechanisms. Here we show that Nmu-deficient (Nmu-/-) mice have high bone mass owing to an increase in bone formation; this is more prominent in male mice than female mice. Physiological and cell-based assays indicate that NMU acts in the central nervous system, rather than directly on bone cells, to regulate bone remodeling. Notably, leptin- or sympathetic nervous system-mediated inhibition of bone formation was abolished in Nmu-/- mice, which show an altered bone expression of molecular clock genes (mediators of the inhibition of bone formation by leptin). Moreover, treatment of wild-type mice with a natural agonist for the NMU receptor decreased bone mass. Collectively, these results suggest that NMU may be the first central mediator of leptin-dependent regulation of bone mass identified to date. Given the existence of inhibitors and activators of NMU action, our results may influence the treatment of diseases involving low bone mass, such as osteoporosis.     

Sympathetic neural influence on bone metabolism in microgravity (Review)

Bone loss is one of the most important complications for astronauts who are exposed to long-term microgravity in space and also for bedridden elderly people. Recent studies have indicated that the sympathetic nervous system plays a role in bone metabolism. This paper reviews findings concerning with sympathetic influences on bone metabolism to hypothesize the mechanism how sympathetic neural functions are related to bone loss in microgravity. Animal studies have suggested that leptin stimulates hypothalamus increasing sympathetic outflow to bone and enhances bone resorption through noradrenaline and β-adrenoreceptors in bone. In humans, even though there have been some controversial findings, use of β-adrenoblockers has been reported to be beneficial for prevention of osteoporosis and bone fracture. On the other hand, microneurographically-recorded sympathetic nerve activity was enhanced by exposure to microgravity in space as well as dry immersion or long-term bed rest to simulate microgravity. The same sympathetic activity became higher in elderly people whose bone mass becomes generally reduced. Our recent findings indicated a significant correlation between muscle sympathetic nerve activity and urinary deoxypyridinoline as a specific marker measuring bone resorption. Based on these findings we would like to propose a following hypothesis concerning the sympathetic involvement in the mechanism of bone loss in microgravity: An exposure to prolonged microgravity may enhance sympathetic neural traffic not only to muscle but also to bone. This sympathetic enhancement increases plasma noradrenaline level and inhibits osteogenesis and facilitates bone resorption through β-adrenoreceptors in bone to facilitate bone resorption to reduce bone mass. The use of β-adrenoblockers to prevent bone loss in microgravity may be reasonable.     

Central infusion of leptin improves insulin resistance and suppresses β-cell function,but not β-cell mass,primarily through the sympathetic nervous system in a type 2 diabetic rat model

AimsWe investigated whether hypothalamic leptin alters β-cell function and mass directly via the sympathetic nervous system (SNS) or indirectly as the result of altered insulin resistant states.Main methodsThe 90% pancreatectomized male Sprague Dawley rats had sympathectomy into the pancreas by applying phenol into the descending aorta (SNSX) or its sham operation (Sham). Each group was divided into two sections, receiving either leptin at 300 ng/kg bw/h or artificial cerebrospinal fluid (aCSF) via intracerebroventricular (ICV) infusion for 3 h as a short-term study. After finishing the infusion study, ICV leptin (3 μg/kg bw/day) or ICV aCSF (control) was infused in rats fed 30 energy % fat diets by osmotic pump for 4 weeks. At the end of the long-term study, glucose-stimulated insulin secretion and islet morphometry were analyzed.Key findingsAcute ICV leptin administration in Sham rats, but not in SNSX rats, suppressed the first- and second-phase insulin secretion at hyperglycemic clamp by about 48% compared to the control. Regardless of SNSX, the 4-week administration of ICV leptin improved glucose tolerance during oral glucose tolerance tests and insulin sensitivity at hyperglycemic clamp, compared to the control, while it suppressed second-phase insulin secretion in Sham rats but not in SNSX rats. However, the pancreatic β-cell area and mass were not affected by leptin and SNSX, though ICV leptin decreased individual β-cell size and concomitantly increased β-cell apoptosis in Sham rats.SignificanceLeptin directly decreases insulin secretion capacity mainly through the activation of SNS without modulating pancreatic β-cell mass.     

Understanding leptin-dependent regulation of skeletal homeostasis

Despite growing evidence for adipose tissue regulation of bone mass, the role of the adipokine leptin in bone remodeling remains controversial. The majority of in vitro studies suggest leptin enhances osteoblastic proliferation and differentiation while inhibiting adipogenic differentiation from marrow stromal cells. Alternatively, some evidence demonstrates either no effect or a pro-apoptotic action of leptin on stromal cells. Similarly, in vivo work has demonstrated both positive and negative effects of leptin on bone mass. Most of the literature supports the idea that leptin suppresses bone mass by acting in the brainstem to reduce serotonin-dependent sympathetic signaling from the ventromedial hypothalamus to bone. However, other studies have found partly or entirely contrasting actions of leptin. Recently one study found a significant effect of surgery alone with intracerebroventricular administration of leptin, a technique crucial for understanding centrally-mediated leptin regulation of bone. Thus, two mainstream hypotheses for the role of leptin on bone emerge: 1) direct regulation through increased osteoblast proliferation and differentiation and 2) indirect suppression of bone formation through a hypothalamic relay. At the present time, it remains unclear whether these effects are relevant in only extreme circumstances (i.e. models with complete deficiency) or play an important homeostatic role in the regulation of peak bone acquisition and skeletal remodeling. Ultimately, determining the actions of leptin on the skeleton will be critical for understanding how the obesity epidemic may be impacting the prevalence of osteoporosis.     

Leptin is a potent stimulator of bone growth in ob/ob mice

Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. The lack of leptin in ob/ob mice, who are homozygous for the obese gene, results in hyperglycemia, hyperinsulinemia, hyperphagia, obesity, infertility, decreased brain size and decreased stature. To this end, we investigated the role of leptin as a hormonal regulator of bone growth. Leptin administration led to a significant increase in femoral length, total body bone area, bone mineral content and bone density in ob/ob mice as compared to vehicle treated controls. The increase in total body bone mass was a result of an increase in both trabecular and cortical bone mass. These results suggest that the decreased stature of the ob/ob mouse is due to a developmental defect that is readily reversible upon leptin administration. Our demonstration that the signalling or long form (Ob-Rb) of the leptin receptor is present in both primary adult osteoblasts and chondrocytes suggests that the growth promoting effects of leptin could be direct. In summary, these results indicate a novel role for leptin in skeletal bone growth and development.     

Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass

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.     

Action of leptin on bone and its relationship to menopause

Backround: 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.     

The molecular clock mediates leptin-regulated bone formation

The hormone leptin is a regulator of bone remodeling, a homeostatic function maintaining bone mass constant. Mice lacking molecular-clock components (Per and Cry), or lacking Per genes in osteoblasts, display high bone mass, suggesting that bone remodeling may also be subject to circadian regulation. Moreover, Per-deficient mice experience a paradoxical increase in bone mass following leptin intracerebroventricular infusion. Thus, clock genes may mediate the leptin-dependent sympathetic regulation of bone formation. We show that expression of clock genes in osteoblasts is regulated by the sympathetic nervous system and leptin. Clock genes mediate the antiproliferative function of sympathetic signaling by inhibiting G1 cyclin expression. Partially antagonizing this inhibitory loop, leptin also upregulates AP-1 gene expression, which promotes cyclin D1 expression, osteoblast proliferation, and bone formation. Thus, leptin determines the extent of bone formation by modulating, via sympathetic signaling, osteoblast proliferation through two antagonistic pathways, one of which involves the molecular clock.     

Effects of increased hypothalamic leptin gene expression on ovariectomy-induced bone loss in rats

Estrogen deficiency results in accelerated bone turnover with a net increase in bone resorption. Subcutaneous administration of leptin attenuates bone loss in ovariectomized (ovx) rats by reducing bone resorption. However, in addition to its direct beneficial effects, leptin has been reported to have indirect (central nervous system-mediated) antiosteogenic effects on bone, which may limit the efficacy of elevated serum leptin to prevent estrogen deficiency-associated bone loss. The present study evaluated the long-term effects of increased hypothalamic leptin transgene expression, using recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy, on bone mass, architecture, and cellular endpoints in sexually mature ovx Sprague-Dawley rats. Ovx rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained for 10 weeks. Additional controls consisted of ovary-intact rats and ovx rats pair-fed to rAAV-Lep rats. Lumbar vertebrae were analyzed by micro-computed tomography and tibiae by histomorphometry. Cancellous bone volume was lower and osteoclast perimeter, osteoblast perimeter, and bone marrow adipocyte density were greater in ovx rats compared to ovary-intact controls. In contrast, differences among ovx groups were not detected for any endpoint evaluated. In conclusion, whereas estrogen deficiency resulted in marked cancellous osteopenia, increased bone turnover and marrow adiposity, increasing hypothalamic leptin transgene expression in ovx rats had neither detrimental nor beneficial effects on bone mass, architecture, or cellular endpoints. These findings demonstrate that the antiresorptive effects of subcutaneous leptin administration in ovx rats are mediated through leptin targets in the periphery.     

Leptin regulates bone formation via the sympathetic nervous system

We previously showed that leptin inhibits bone formation by an undefined mechanism. Here, we show that hypothalamic leptin-dependent antiosteogenic and anorexigenic networks differ, and that the peripheral mediators of leptin antiosteogenic function appear to be neuronal. Neuropeptides mediating leptin anorexigenic function do not affect bone formation. Leptin deficiency results in low sympathetic tone, and genetic or pharmacological ablation of adrenergic signaling leads to a leptin-resistant high bone mass. beta-adrenergic receptors on osteoblasts regulate their proliferation, and a beta-adrenergic agonist decreases bone mass in leptin-deficient and wild-type mice while a beta-adrenergic antagonist increases bone mass in wild-type and ovariectomized mice. None of these manipulations affects body weight. This study demonstrates a leptin-dependent neuronal regulation of bone formation with potential therapeutic implications for osteoporosis.     

Plasma adiponectin concentration in healthy pre- and postmenopausal women: relationship with body composition, bone mineral, and metabolic variables

The aim of the current investigation was to determine the possible relationships of fasting adiponectin level with body composition, bone mineral, insulin sensitivity, leptin, and cardiorespiratory fitness parameters in 153 women. Subjects were classified as premenopausal (n = 42; 40.8 +/- 5.7 yr) if they had regular menstrual periods, early postmenopausal (n = 49; 56.7 +/- 3.6 yr) if they had been postmenopausal for more than >1 yr but <7 yr (5.5 +/- 1.3 yr), and postmenopausal (n = 62; 72.2 +/- 4.5 yr) if they had been postmenopausal for >7 yr. All women studied had a body mass index (BMI) <30 kg/m(2). Adiponectin values were higher (P < 0.05) in middle-aged (12.0 +/- 5.1 microg/ml) and older (15.3 +/- 7.3 microg/ml) postmenopausal women compared with middle-aged premenopausal women (8.4 +/- 3.2 microg/ml). Mean plasma adiponectin concentration in the total group of women (n = 153) was 12.2 +/- 6.3 microg/ml and was positively related (P < 0.05) to age, indexes of overall obesity (BMI, body fat mass), and cardiorespiratory fitness (PWC) values. In addition, a negative association (P < 0.05) between adiponectin with central obesity (waist-to-hip and waist-to-thigh ratio), fat-free mass, bone mineral (bone mineral content, total and lumbar spine bone mineral density), and leptin and insulin resistance (insulin, fasting insulin resistance index) values was observed. However, multivariate regression analysis revealed that only age, fasting insulin resistance index, and leptin were independent predictors of adiponectin concentration. In conclusion, circulating adiponectin concentrations increase with age in normal-weight middle-aged and older women. It appears that adiponectin is independently related to age, leptin, and insulin resistance values in women across the age span and menstrual status.     

Preactivation of β-catenin in osteoblasts improves the osteoanabolic effect of PTH in type 1 diabetic mice

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1–3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.     

Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers

Binding of leptin to the leptin receptor is crucial for body weight and bone mass regulation in mammals. Leptin receptors were shown to exist as dimers, but the role of dimerization in receptor activation remains unknown. Using a quantitative Bioluminescence Resonance Energy Transfer approach, we show here in living cells that approximately 60% of the leptin receptor exists as constitutive dimers at physiological expression levels in the absence of leptin. No further increase in leptin receptor dimerization was detected in the presence of leptin. Importantly, in cells expressing the short leptin receptor isoform, leptin promoted a robust enhancement of energy transfer signals that reflect specific conformational changes of pre-existing leptin receptor dimers and that may be used as read-out in screening assays for leptin receptor ligands. Both leptin receptor dimerization and the leptin-induced energy transfer were Janus kinase 2-independent. Taken together, our data support a receptor activation model based on ligand-induced conformational changes rather than ligand-induced dimerization.     

Increased bone mineral density and serum leptin in non-obese girls with precocious pubarche: relation to low birthweight and hyperinsulinism.

BACKGROUND: Hyperinsulinism and hyperandrogenism have the capacity to increase bone mineral density (BMD) and serum leptin, independently of body fat mass. We therefore assessed lumbar BMD and serum leptin in girls with the sequence of a low birthweight and precocious pubarche (PP) in childhood, in whom hyperinsulinism and hyperandrogenism have been described. METHODS: Fifty-two non-obese PP girls were studied (age range 6.9-14.9 years). Serum leptin was also measured in 42 control girls, matched for age, body mass index and pubertal stage. RESULTS: BMD SDS, measured by dual-energy X-ray absorptiometry, was elevated in PP girls compared to the population reference (0.39 +/- 0.18 SDS; p = 0.03) and bone age, assessed from hand radiographs, was significantly advanced compared to chronological age (1.2 +/- 0.1 years; p < 0.0005). CONCLUSION: Compared to control girls, PP girls had higher leptin levels for degree of body mass index (PP girls: 9.4 +/- 0.6 ng/ml; controls: 7.8 +/- 0.6 ng/ml; p = 0.01). In PP girls, serum leptin was inversely related to birthweight (r = -0.32, p = 0.01) and positively related to free androgen index (FAI) (r = 0.71, p < 0.0005). BMD SDS was also inversely related to birthweight (r = -0.26, p < 0.05) and positively related to serum leptin (r = 0.42, p < 0.05), FAI (r = 0.45, p < 0.05) and mean serum insulin during oral glucose tolerance testing (MSI) (r = 0.59, p < 0.0005). In multiple regression, MSI was the strongest determinant of BMD SDS (beta = 0.50, p = 0.002). In conclusion, elevated BMD and serum leptin in non-obese PP girls were related to degrees of low birthweight, hyperinsulinism and hyperandrogenism. The characteristic hyperinsulinism of PP girls is proposed to be the key variable in this constellation.     

Diet‐induced Obese Mice Are Leptin Insufficient After Weight Reduction

Behavioral therapies aimed at reducing excess body fat result in limited fat loss after dieting. To understand the causes for maintenance of adiposity, high‐fat (HF) diet–induced obese (DIO) mice were switched to a low‐fat chow diet, and the effects of chow on histological and molecular alterations of adipose tissue and metabolic parameters were examined. DIO mice reduced and stabilized their body weights after being switched to chow (HF‐chow), but retained a greater amount of adiposity than chow‐fed mice. Reduction in adipocyte volume, not number, caused a decrease in fat mass. HF‐chow mice showed normalized circulating insulin and leptin levels, improved glucose tolerance, and reduced inflammatory status in white adipose tissue (WAT). Circulating leptin levels corrected for fat mass were lower in HF‐chow mice. Leptin administration was used to test whether reduced leptin level of HF‐chow mice inhibited further fat loss. Leptin treatment led to an additional reduction in adiposity. Finally, HF‐HF mice had lower mRNA levels of β₃ adrenergic receptor (β₃‐AR) in epididymal WAT (EWAT) compared to chow‐fed mice, and diet change led to an increase in the WAT β₃‐AR mRNA levels that were similar to the levels of chow‐fed mice, suggesting an elevation in sympathetic activation of WAT during diet switch relative to HF‐HF mice leading to the reduced leptin level and proinflammatory cytokine content. In summary, HF‐chow mice were resistant to further fat loss due to leptin insufficiency. Diet alteration from HF to low fat improved metabolic state of DIO mice, although their adiposity was defended at a higher level.     

Circadian variation of serum leptin in healthy and diabetic men

Leptin, from the Greek leptos, meaning thin (in reference to its ability to reduce body fat stores), is a hormone secreted primarily by adipocytes. At one time, leptin was portrayed as a potential means of combating obesity. Recently, leptin has been identified as a potent inhibitor of bone formation, acting through the central nervous system. Since numerous studies clearly show that bone remodeling is circadian rhythmic with peak activity during sleep, it is of interest to explore circadian variability in serum leptin. Accordingly, circadian characteristics of serum leptin were examined in 7 clinically healthy men and 4 obese men with type II diabetes. Blood samples were collected for 24 h at 3 h intervals beginning at 19:00. The dark (sleep) phase of the light-dark cycle extended from 22:30 to 06:30, with brief awakening for sampling at 01:00 and 04:00. Subjects consumed general hospital meals (2400 calories) at 16:30, 07:30, and 13:30. Serum leptin levels were determined by a R&D Systems enzyme immunoassay technique. Data were analyzed by linear least-squares estimation using the population multiple components method. A statistically significant (P < .018) circadian rhythm modeled by a single 24 h cosine curve characterized the data of each group. The 24 h mean leptin level was statistically greater (P < .001) in the obese diabetic men than in the healthy men (9.47 +/- 0.66 ng/mL vs. 24.07 +/- 1.71 ng/mL, respectively). Higher leptin levels occurred between midnight and roughly 02:30, and lowest leptin levels occurred between noon and the early afternoon. The phasing of this rhythm is similar to the circadian rhythm in bone remodeling previously described. Our results suggest the findings from a single morning blood sampling for leptin may be misleading since it may underestimate the mean 24 h and peak concentrations of the hormone.