PLoS One. 2016 Feb 23;11(2):e0150102. doi: 10.1371/journal.pone.0150102.
Constitutively Elevated Blood Serotonin Is Associated With Bone Loss and Type 2 Diabetes in Rats
Authors: Igor Erjavec1¶, Tatjana Bordukalo-Niksic1¶, Jelena Brkljacic1, Danka Grcevic2, Gordana Mokrovic3, Maja Kesic3, Dunja Rogic4, William Zavadoski5, Vishwas M Paralkar5, Lovorka Grgurevic1, Vladimir Trkulja6, Lipa Cicin-Sain3 and Slobodan Vukicevic1*
1Laboratory of Mineralized Tissues, Center for Translational and Clinical Research, School of Medicine, University of Zagreb, Croatia.
2Department of Physiology and Immunology, School of Medicine, University of Zagreb, Croatia.
3Laboratory for Neurochemistry and Molecular Neurobiology, Molecular Biology Department, Rudjer Boskovic Institute, Zagreb, Croatia.
4Clinical Hospital Center Zagreb, Croatia.
5Karos Pharmaceuticals, New Haven, USA.
6Department of Pharmacology, School of Medicine, University of Zagreb, Croatia.
¶ These authors contributed equally to this work.
Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.
Serotonin (5-hydroxytryptamine, 5HT) has multiple functions in peripheral organs acting via 15 transmembrane receptors. The impact of serotonin on bone metabolism is the subject of many recent studies; however, without unequivocal conclusion. Thus, in one study reduced serotonin in mice lacking TPH1, the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton with high osteoblastic activity , while in other studies Tph1 deletion led to either an initial increase in bone mineral density (BMD) due to inhibition of osteoclastic bone resorption  or had no bone effect . In women, it has been shown that circulating 5HT inversely associates with the body and spine areal BMD  ; however, other study found positive association between serum 5HT and bone parameters in postmenopausal women . Use of antidepressant medications, which act on serotonin system, has been shown to have impact on decreasing BMD and to increase risk for osteoporosis . The role of 5HT in bone metabolism remains therefore poorly understood.
Here, we developed a novel rodent model of constitutionally altered 5HT homeostasis established by crossing Wistar rats selected for high or low platelet 5HT level (PSL) and the activity of platelet 5HT transporter, which regulates the uptake of 5HT from blood plasma into platelets. Selective breeding through several successive generations of animals resulted in two sublines of rats with constitutional hyper- or hyposerotoninemia and constitutionally altered serotonin homeostasis in general, named high-5HT and low-5HT subline . We hypothesized that these constitutive differences in serotonergic phenotype might affect skeletal tissue. In our study we demonstrated that rats with high PSL had an increased bone turnover with subsequent bone loss, but also developed a type 2 diabetes (T2D) phenotype.
In all experiments rats from high-5HT and low-5HT sublines differed by approximately 2-fold in their platelet serotonin level (PSL) and platelet serotonin uptake. High-5HT rats also demonstrated consistently higher values of body weight and femur length between the age of 2 and 12 months, and progressively decreased muscle strength, as assessed by the string test.
Bone analyses, performed by microCT, showed lower BV/TV ratio and lower number of trabecules at lumbar spine and distal femur in high-5HT rats. Differences between high-5HT and low-5HT subline became more pronounced with ageing. Bone histomorphometric analysis confirmed results of microCT and revealed an increased osteoblast activity via mineral apposition rate (MAR) and bone formation rate (BFR) in young high-5HT rats.
To further explore the relationship between 5HT and bone loss, we measured the bone remodeling markers and related calciotropic hormones in plasma. High-5HT rats had higher plasma C-telopeptide level and lower FGF23 at younger age (2 months old) and lower 1,25(OH)2D3 at older age (12 monhts old). With ageing, osteocalcin decreased and C-telopeptide increased in both sublines, which confirmed the morphometric analyses.
Measurements of glucose and insulin in plasma of high-5HT and low-5HT animals of different age revealed that, over time, high 5HT rats developed higher plasma glucose and insulin levels, which resembled the type 2 diabetes phenotype. Glucose intolerance and insulin resistance in high-5HT subline was evidenced by functional tests (glucose and insulin tolerance test) performed at 6 months of age. In addition, with ageing, high-5HT rats showed more pronounced increased total serum cholesterol and visceral fat, as well as an increased number and size of b-cell pancreatic islets.
Although the exact etiology of the T2D phenotype in the high-5HT rats is unclear, 5HT has already been reported to affect β cell mass and function, for example, during pregnancy in otherwise healthy animals . 5HT has also been shown to modulate insulin secretion from pancreatic β cells via serotonylation of GTPases , further supporting the finding that elevated 5HT increases insulin release in high-5HT rats. Although the precise sequence of events that occurs in bone following the development of insulin resistance has not been resolved, literature evidence demonstrates that patients with T2D have lower serum bone formation markers, decreased muscle tone and reduced bone strength .
To further explore the relationship between 5HT, insulin and calciotropic hormones, we treated animals from both sublines with PTH, 1,25(OH)2D3 or fluvoxamine, a 5HT re-uptake inhibitor. As expected, fluvoxamine lowered PSL in both sublines. However, this was paralleled by a drop in plasma insulin in low-5HT, but not in high-5HT animals. Treatment with PTH and 1,25(OH)2D3 increased serum calcium in both sublines, but, interestingly, in high-5HT rats, this was paralleled by a PSL increase in response to PTH (which caused a modest increase in serum calcium) and a decrease in response to 1,25(OH)2D3 (which caused a profound increase in serum calcium). Additionally, a single injection of streptozotocin in high-5HT rats, which reduced plasma insulin level, resulted also in a significant decrease in PSL. This novel association between PSL and plasma insulin was additionally confirmed by correlation analysis between these two parameters.
In an additional experiment, 12 months old high-5HT rats received LX1032, an TPH1-specific inhibitor, which was expected to reduce 5HT synthesis in the gut and consequently PSL. Treatment of 36 days reduced PSL in treated animals, which was parallelled by an increased bone volume and trabecular number, as well as by decreased trabecular separation. This experiment showed that inhibition of TPH1, and therefore decrease of circulatory 5HT level, can improve bone status.
Collectively, increased PSL in rats is associated with a complex endocrine, paracrine and autocrine activities of various cells and organs leading to hypertrophy of β-pancreatic islets, increased plasma glucose and lipids and decreased 1,25(OH)2D3 that at least in part contributed to the increased remodeling rate and bone loss. Treatment of high- and low-5HT rats with PTH, 1,25(OH)2D3, SSRI and streptozotocin influenced PSL, but also plasma insulin, both directly and via changes of serum calcium concentration. The results of our experiments also suggested that bone phenotype deduced from 5HT deficient mice cannot be directly translated into high-5HT PSL phenotype in rats. In conclusion, our findings suggested an association between the increased PSL, T2D phenotype and bone loss in a complex systemic environment composed of unbalanced calciotropic hormones and calcium influence on the 5HT metabolism.
Figure 1. Schematic representation of the systemic role of peripheral serotonin (5HT).The quantityof 5HT in platelets depends on the serotonin transporter (5HTT). Elevated 5HT bidirectionally influences the plasma insulin level and induces the hyperthrophy of pancreatic β-cells, leading with ageing to type 2 diabetes phenotype. Increased insulin and 5HT have an additive effect on bone formation (osteoblast activity).Elevated 5HT increases both bone formation and resorption (osteoclast activity), thus increasing the bone turnover and resulting in the net bone loss. Inset: Wistar rat 5HT-sublines developed by selective breeding from the base population. The sublines differ in platelet 5HT level (PSL) and platelet 5HT uptake (PSU) by approximately 2-fold after several generation of selective breeding.
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