Marine alga Sargassum horneri active component: Prevention of obese diabetic bone loss

Osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public heath problem. Food and nutritional factors may play a role in the prevention of bone loss with aging. This, however, is poorly understood. Among marine algae of Undaria pinnatifida, Sargassum horneri, Eisenia bicyclis, Cryptonemia scmitziana, Gelidium amansii, and Ulva pertusa Kjellman that were gathered seasonally, Sargassum horneri (S. horneri) was found to reveal a unique anabolic effect on bone. S. horneri active component revealed a stimulatory effect on osteoblastic bone formation and a suppressive effect on osteoclastic bone resorption in vitro, thereby increasing bone mass. Moreover, S. horneri active component suppressed adipogenesis in bone marrow cells in vitro. Intake of S. horneri active component revealed a preventive effect on bone loss in animal models for type 1 diabetes. Moreover, intake of S. horneri active component was demonstrated to reveal anabolic effects on bone metabolism in healthy human. S. horneri active component, a new osteogenic factor, may be a useful in the improvement of obese diabetic osteoporosis.


Introduction
Bone is a dynamic tissue that preserves skeletal size, shape, and structural integrity and regulates mineral homeostasis. Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption. Aging and numerous pathological processes induce decrease in bone formation and increase in bone resorption, leading to osteoporosis that is a devastating bone disease [1]. Osteoporosis, which is induced with increase in bone loss, is widely recognized as a major public health problem. The most dramatic expression of the disease is represented by fractures of the proximal femur for which the number increases as the population ages [2].
Currently, obesity and diabetes are a major health problem worldwide with growing in prevalence. Obesity and diabetes induce secondary diseases with various pathophysiologic states including cardiovascular disease, neural disturbance, kidney disease, cancer and osteoporosis. Osteoporosis associated with obesity and diabetes has also been noticed [3][4][5][6]. Diabetes is seen frequent in the elderly and therefore frequently coexists with osteoporosis. The incidence of metabolic disease, including obesity and obese type 2 diabetes, has been increased to epidemic levels in recent years [5]. Type 1 and obese type 2 diabetes have been associated with increased fracture risk [4]. Thus, obesity, diabetes and osteoporosis are closely related. One of the shared features is that osteoblasts and adipocytes differentiate from a common precursor cell in the bone marrow mesenchymal stem cells [7,8]. There is an inverse relationship between differentiation of mesenchymal stem cells to osteoblasts and adipocytes [7,8]. It may be important to prevent and improve obese diabetic osteoporosis in clinical aspects.
There is growing evidence that the supplementation of nutritional and food factors may have preventive effects on bone loss that is induced in animal model of osteoporosis and in human subjects [9][10][11][12]. Functional factors in food and plants, which regulate on bone homeostasis, have been to be worthy of notice in maintaining of bone health and prevention of bone loss with increasing age [13][14][15]. The effect of marine algae on bone metabolism is poorly understood.
Undaria pinnatifida, Sargassum horneri, Eisenia bicyclis, Cryptonemia scmitziana, Gelidium amansii, and Ulva pertusa Kjellman are applied as food materials. Among these marine algae, Sargassum horneri (S. horneri) was found to have a unique anabolic effect on bone [16]. S. horneri extract has been found to have a stimulatory effect on osteoblastic bone formation and a suppressive effect on osteoclastic bone resorption in vitro, thereby increasing bone mass. Moreover, S. horneri active component was found to reveal suppressive effects on adipogenesis in bone marrow cells [17]. Intake of S. horneri active component revealed a preventive effect on bone loss in animal models for type 1 diabetes [18]. This review has been written to outline our recent advances that have been made concerning preventive effects of S. horneri active component on osteoporosis.

Bone homeostasis and osteoporosis
Bone metabolism plays a physiological role to maintain skeletal structure and to regulate mineral homeostasis. Bone remodeling and modeling underpin development and maintenance of the skeletal system [19]. Bone modeling is responsible for growth and mechanically induced adaptation of bone, and it requires that the processes of bone formation and bone removal (resorption). Process of bone remodeling that makes bone unique among organs and tissues, and add so many levels of complexity with respect to interactions along remodeling sequence by systemic influences (hormones), stress action (physical activity/weight bearing), and growth factors and cytokines produced from the bone cells or factors that come from nearby cells in the Integr Food Nutr Metab, 2014 doi: 10.15761/IFNM.1000101 marrow tissues. Bone homeostasis, which maintains bone mass, is skillfully regulated through a delicate balance between osteoblastic bone formation and osteoclastic bone resorption [20][21][22]. Bone acts as major storage site for growth factors, which are produced by osteoblasts, diffuse into newly deposited osteoid and are stored in the bone matrix including isulin-like growth factors (IGF-I and II), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF), or bone morphologic proteins (BMPs) [23,24]. These bone-derived factors, which can be liberated during subsequent periods of bone resorption, act in an autocrine, paracrine, or delayed paracrine fashion in the local microenvironment of the bone surface.
Osteoporosis is a disease characterized by loss in bone density and bone strength and deterioration of bone microarchitecture, resulting in increased risk for bone fractures. The most dramatic expression of osteoporosis is represented by fractures of the proximal femur for which the number increases as the population ages [2]. Osteoporosis is a common metabolic disease and generally affects people at an advanced age and suffering from other chronic diseases. It is more common in women and a significant loss of bone mass after menopause begins. Bone mass is dramatically reduced after menopause, which depresses secretion of ovarian hormone (estrogen) in women [1]. Deficiency of estrogen advances osteoclastic bone resorption. This is very important as a primary osteoporosis. Postmenopausal osteoporosis is the archetypal osteoporotic condition in women after menopause. Osteoporosis is a major cause of increased morbidity and mortality affecting the aging population. It has been estimated that osteoporosis affects at least 200 million women worldwide, one third of women aged between 60 and 70 years and two thirds over 80 years [2,25]. In 1995, incidence of osteoporotic fractures in the U.S. was about 1.5 million, of which 750 000 vertebral fractures, 250,000 hip fractures, 250,000 fractures in the wrist fracture and 250 000 other locations. According to a recent World Health Organization report, osteoporosis has become a global health problem with a disease incidence and mortality rate similar to that of cardiovascular diseases, cancer and diabetes [3][4][5][6]. Osteoporosisis is widely recognized as a major public health threat.

S. horneri active component stimulates osteoblastic bone formation
Marine algae S. horneri [Sargassum horneri (Turner) C. Agardh] was seasonally gathered from the coast at Shimoda (Shizuoka Prefecture, Japan) and Miyako (Iwate Prefecture, Japan), and it was freeze-dried and powered [16]. The gathered fresh marine algae were homogenized in distilled water and are centrifuged at 5500 g in a refrigerated centrifuge for 10 minutes. The 5500 g supernatant fraction was pooled for freeze-drying. The powder of the water-solubilized extract extract was dissolved in ice-cold distilled water for use in the experiments. The water-solubilized extract from S. horneri was purified through the method of membrane fractionation to collect active component of various molecular weights.
The direct effect of S. horneri active component on bone formation and mineralization has been examined using bone tissues and MC3T3 preosteoblastic cells in vitro [26,27]. Preosteoblastic cells were differentiated into mineralizing osteoblasts in the presence of S. horneri active component in culture with mineralizing medium for 21 days in vitro [27]. S. horneri active component (10 and 25 μg/ml) was found to potently enhance mineralization in both MC3T3 cells [27]. The S. horneri extract (25-100 μg/ml of medium) did not have a significant effect on cell number of MC3T3-E1 cells with culture [27]. S. horneri active component did not appear to mediate direct toxic effects on the cultures as cells were proliferated robustly over the culture period, and were still alive and visibly attached to the plate at the end of the experiment [27].
Bone morphogenic proteins (BMPs) such as BMP-2 are anabolic agents that signal through the Smad signaling pathway [24]. The effect of S. horneri extract on basal and BMP-2-induced Smad activation has been shown using a Smad 4-luciferase reporter responsive to all Smad species [27]. S. horneri active component did not have a direct effect on basal Smad activation. S. horneri active component (50 or 100 μg/ ml) enhanced Smad-activation induced by BMP-2. Also, S. horneri active component (25 -100 μg/ml) enhanced Smad-activation induced by TGF-β1 [27]. NF-κB activation is a potent inhibitory to osteoblast differentiation, and TNF-α-induced NF-κB activation leads to Smad suppression in MC3T3 osteoblast precursors [28,29]. Whether S. horneri active component is able to prevent TNF-α-induced NF-κB activation in MC3T3 osteoblast precursors was examined [27]. S. horneri active component (25-100 μg/ml of medium) prevented TNFα-induced NF-κB activation, although S. horneri active component did not have a direct effect on basal NF-κB activation. Thus, S. horneri active component has been shown to stimulate osteoblastic differentiation and mineralization in vitro. TGF-β1-and BMP-2-induced activation of Smad signaling respectively plays an important role in the early commitment and differentiation of osteoblasts [30]. S. horneri active component was found to enhance BMP-2-or TGF-β1-induced Smad activation with the dose-dependency [27]. S. horneri extract-induced Smad enhancement may be an important to stimulate osteoblastic differentiation and mineralization.
The anabolic effect of S. horneri active component on bone tissues in vitro has been found. Rat femoral-metaphyseal tissues were cultured in a medium containing water-solubilized extract (25 and 50 μg/ml) obtained from U. pinnatifida, S. horneri, E. bicyclis, or C. scmitziana in vitro [16,26]. Bone calcium content was significantly elevated in the presence of S. horneri active component (25 and 50 μg/ml). No effect was seen in the extracts of other marine algae. In addition, water suspensions (5%) of marine alga powder were orally administered once daily for 7 days. Bone calcium content was significantly increased after the administration of U. pinnatifida, S. horneri, E. bicyclis, or C. scmitziana [16]. Also, bone alkaline phosphatase activity, which is an enzyme for calcification [32], was significantly enhanced with the administration of S. horneri or G. amansii. Thus, S. horneri active component has a unique anabolic effect on bone calcification in vitro and in vivo [16]. The effects of S. horneri active component in increasing calcium content, alkaline phosphatase activity, and deoxyribonucleic acid (DNA) content in the femoral-diaphyseal and Integr Food Nutr Metab, 2014 doi: 10.15761/IFNM.1000101 -metaphyseal tissues in vitro was completely abolished in the presence of cycloheximide, an inhibitor of protein synthesis [16]. Anabolic effect of S. horneri active component may be resulted from newly synthesized protein components.

S. horneri active component suppresses osteoclastic bone resorption
S. horneri extract has been shown to suppress osteoclastic bone resorption. To examine the effect of S. horneri active component on osteoclast formation, RAW264.7 osteoclast precursors were differentiated into mature osteoclasts by stimulation with RANKL in the presence or absence of S. horneri active component with the dose range of 5 to 100 μg/ml of medium [27]. The S. horneri active component did not have effects on cell number of preosteoclasts (RAW267.4 cells) with culture. S. horneri extract (100 μg/ml) did not have a toxicity to the precursors suppressing their proliferation over 7 days of culture [27]. S. horneri active component (25-100 μg/ml) significantly suppressed osteoclast formation induced by RANKL [27].
The NF-κB signal transduction pathway is essential for the generation of osteoclasts [20,21]. The effect of S. horneri active component on NF-κB activation by RANKL in osteoclast precursors has been examined. RAW 264.7 cells were transfected with an NF-κB reporter and stimulated with RANKL to induce NF-κB activity in the presence or absence of S. horneri active component (5-100 μg/ml) [27]. S. horneri active component did not have a significant effect on basal NF-κB activity [27]. However, RANKL-induced increase in NF-κB activity was significantly blunted in the presence of S. horneri active component [27].
S. horneri active component with dose-dependency, which did not have cell toxicity, was found to have a suppressive effect on RANKLstimulated osteoclastogenesis [27]. RANKL, the key osteoclastogenic cytokine, is central to formation of osteoclasts, the cells that resorb bone, through NF-κB signaling [20,21]. S. horneri active component suppressed RANKL-induced NF-κB activation in osteoclast precursor cells [27]. This finding coincided with the observation that S. horneri extract suppresses RANKL-induced osteoclastogenesis [27]. S. horneri active component may suppress osteoclastogenesis through suppression of NF-κB activation.
The effect of S. horneri active component on mineralization in MC3T3-E1 cells with culture for 21 days was observed at 10 μg/ml [27]. However, the effects of S. horneri active component on Smad activity in MC3T3-E1 cells and on NF-κB activity in RAW 264.7 cells with culture for 24 hours were observed at 25 μg/ml [27]. The effects of S. horneri active component on osteoclastogenesis in RAW 264.7 cells with culture for 6 days were also revealed at 25 μg/ml [27]. The effect of S. horneri active component on osteoblastogenesis and osteoclastogenesis may be observed with lower doses in the culture for longer periods.
Suppressive effects of S. horneri active component on bone resorption using femoral tissues in vitro have been demonstrated [33]. Parathyroid hormone (PTH) and prostagrandine E 2 (PGE 2 ) are known to induce osteoclastic bone resorption [34]. PTH-and PGE 2 -induced decreases in bone calcium content were inhibited after culture with S. horneri active component (10,25, and 50 μg/ml) [33]. Also, S. horneri active component suppressed PTH-or PGE 2 -induced increase in medium glucose consumption and latic acid production by the bone tissues [33]. Moreover, S. horneri active component blocked PTHinduced increase in acid phosphatase activity in the diaphyseal and metaphyseal tissues [33]. These findings indicate that S. horneri active component has a direct suppressive effect on bone resorption in tissue culture in vitro.
S. horneri active component possesses a potent Smad activation and anti-NF-κB activity and may have promise for development into an antiosteoporotic material capable of promoting new bone formation while simultaneously reducing bone resorption. S. horneri active component may be usefulness as a tool in the prevention of osteolysis with various pathophysiologic states.

S. horneri active component suppresses adipogenesis in bone marrow cells
S. horneri active component has been found to reveal suppressive effects on adipogenesis, which is involved in obesity [17]. Mouse bone marrow cells were cultured for 3 days in differentiation medium (containing dexamethasone and 3-isobutyl-1-methylxanthine) with either vehicle or S. horneri active component (5,10,25, and 50 μg/ml of medium per well) for 48 hours, and then the cells were cultured in the presence of insulin or S. horneri active component (5,10,25, and 50 μg/ ml of medium per well) for an additional 4 days [17]. Adipogenesis was markedly enhanced after culture with medium containing insulin [17]. This enhancement was clearly suppressed in the presence of S. horneri active component (10-50 μg/ml of medium) [17]. Thus, S. horneri active component was found to cause suppression of adipogenesis in bone marrow culture in vitro. Culture with S. horneri active component (10-50 μg/ml of medium), which was added at early stage of differentiation of bone marrow cells to adipocytes, was also found to cause a significant suppression of adipogenesis in bone marrow culture in vitro [17]. Thus, S.horneri active component was found to suppress processes of the differentiation from bone marrow mesenchymal stem cells to pre-adipocytes and from pre-adipocytes to mature adipocytes. S. horneri active component suppresses adipogenesis and may lead to prevention of obesity. Intake of S. horneri active component may be useful tool in the prevention and therapy on obesity and obesityinduced type 2 diabetes.

Preosteoclasts Osteoclasts
S. horneri active component, which contains less than molecular weight 3000, has been shown to stimulate osteoblastic bone formation and suppress osteoclastic bone resorption in cloned bone cells but not bone marrow cells [27]. S. horneri active component, which contains less than molecular weight 3000, reveals suppressive effects on the differentiation from bone marrow mesenchymal stem cells to adipocytes, thereby inhibiting adipogenesis. S. horneri active component may be useful tool in the prevention and treatment of obesity with enhancement of adipogenesis.

Characterization of active component in S. horneri
S. horneri active component has been found to stimulate osteoblastic bone formation and to suppress osteoclastic bone resorption. Those active components have been found to be present in S. horneri extract obtained from various coasts in both Japan (Shimoda and Iwate, Japan) and China [35]. The active component of S. horneri extract in stimulating bone calcification has been found to be near molecular weight (MW) 3000 [27]. Meanwhile, the active component of S. horneri extract in inhibiting osteoclastic cell formation was less than MW 3000 and over MW 50000 [23,33]. These components were stable under heat treatment. It is speculated that the active component in stimulating bone calcification is a chemical but not peptide, and that the component over MW 50000 in inhibiting bone resorption may be a polysaccharide. These active components obtained from the coasts of Iwate (Japan) or China showed an identical molecular weight. The active component, which stimulates osteoblastogenesis and suppresses osteoclastogenesis, was found to be present in the components less than MW 3000 of S. horneri extract using preosteoblastic cells and RAW267.4 cells in vitro [27]. Active component that stimulates osteoblastic bone formation and suppresses osteoclastic bone resorption may be identical.

S. horneri active component improves diabetic bone loss in vivo
The anabolic effects of S. horneri active component on bone components in the femoral tissues of young and aged rats in vivo have been demonstrated [36]. Calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and -metaphyseal tissues of young male (4-week-old) rats were increased after the administration of S. horneri active component (25,50, and 100 mg/ kg) for 7 days [36]. Moreover, these bone components in the femoraldiaphyseal and -metaphyseal tissues of aged female (50-week-old) rats were increased after the administration of S. horneri active component (100 mg/kg) for 14 days, suggesting a preventive effect on bone loss with increasing age [36].
Intake of S. horneri active component has been shown to reveal a preventive effect on bone loss in a diabetes state [18]. Diabetes has been shown to induce bone loss [4,37,38]. Streptozotocin (STZ) induces decrease in insulin secretion in pancreatic cells and causes type 1 diabetes. The oral administration of S. horneri active component (100 mg/kg body weight) to STZ (60 mg/kg body weight)-diabetic rats was found to have a preventive effect on bone loss with diabetes in vivo [18]. This finding suggests that the dietary intake of S. horneri active component has a preventive effect on bone loss in diabetes state. When the femoral tissues obtained from STZ-diabetic rats were cultured in medium containing S. horneri active component, the femoral calcium content and alkaline phosphatase activity were increased in vitro [18]. Alkaline phosphatase is related to bone calcification [32]. S. horneri active component has a stimulatory effect on bone formation [16,27] and a suppressive effect on bone resorption in vitro [27,33].Thus, suppressive effects of S. horneri active component on diabetes-induced bone loss may be resulted from a stimulatory effect on bone formation and a suppressive effect on bone resorption.
Interestingly, oral administration of S. horneri active component (100 mg/kg body weight) to STZ-diabetic rats was found to reveal preventive effects on the decrease in body weight and the increase in serum glucose and triglyceride levels induced in the diabetic state [18]. This was the first time finding. Intake of S. horneri active component has restorative effects on serum biochemical finding with diabetes in vivo.
Thus, intake of S. horneri active component was demonstrated to reveal preventive effects on bone loss, hyperglycemia and hyperlipidemia in type 1 diabetes [18]. S. horneri active component in preventing bone loss induced with diabetic state may be identical to the component that prevents an elevation of serum glucose and triglyceride levels with diabetes. This remains to be elucidated.

S. horneri active component reveals anabolic effects on bone metanolism in human subjects
Supplemental intake of S. horneri active component has been shown to reveal anabolic effects on bone metabolism in human subjects [39]. This study was undertaken to determine the effect of supplemental intake of S. horneri active component on circulating bone metabolic markers in healthy human [39]. Thirty-six volunteers, aged 20-60 years (16 men and 20 women), were enrolled. Volunteers were divided into three groups; placebo tablet without S. horneri active component (5 men and 7 women), tablet containing S. horneri active component at 300 mg/day (6 men and 7 women) or 900 mg/day (5 men and 6 women) [39]. Placebo or S. horneri active component ablet was ingested once a day for 4 or 8 weeks [39]. The intake of dietary S. horneri active component (900 mg/day) for 8 weeks did not have a significant alteration in other biochemical markers for the metabolic function of organs, suggesting that the intake does not have toxic effects in humans [39].
Bone-specific alkaline phosphatase [40] and γ-carboxylated osteocalcin [41] are serum bone markers of bone formation, and bone tartrate-resistant acid phosphatase (TRACP) [42] and N-telopeptides of type I collagen [43] are markers of bone resorption. Serum bone-specific alkaline phosphatase or γ-carboxylated osteocalcin concentration was not significantly changed after the intake of S. horneri active component (300 or 900 mg/day) for 4 or 8 weeks [39]. Serum bone TRACP activity was significantly decreased after the intake of S. horneri active component (300 or 900 mg/day) for 8 weeks [39]. Serum N-telopeptides of type I collagen concentration was significantly decreased after the intake of S. horneri active component (900 mg/day) for 8 weeks [39]. Meanwhile, serum calcium, inorganic phosphorus, and other biochemical findings were not changed after the intake of S. horneri active component (300 or 900 mg/day) for 4 or 8 weeks [39]. Thus, the prolonged intake of S. horneri active component has inhibitory effects on bone resorption in humans was demonstrated.
S. horneri active component was found to reveal a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. Supplemental intake of S. horneri active component reveals suppressive effects on bone resorption and later may reveal stimulatory effects on bone formation in humans, thereby increasing bone mass. Supplemental intake of S. horneri active component may have preventive and restorative effects on osteoporosis with increasing age and post-menopausal women.

Prospect
Bone loss is induced with increasing aging and numerous pathological processes including cardiovascular disease, neural disturbance, kidney disease, inflammatory states and cancer, leading to osteoporosis, Currently, bone loss with obese diabetes is widely recognized as a major public health problem. Functional food factors may reveal potential effects to improve bone loss with various pathologic states. Among various marine algae that are applied to food, S. horneri active component was found to reveal a unique anabolic effect on bone mass due to suppressing adipogenesis and osteoclastic bone resorption and stimulating osteoblastic bone formation. Supplemental intake of S. horneri active component may be usefulness in the prevention and improvement of bone loss in various pathologic states including obese diabetes. Development of this functional food biomaterial will be expected in clinical fields.