Metabolic reprogramming of osteoclasts represents a therapeutic target during the treatment of osteoporosis

Osteoclasts are specialised bone resorbing cells that control both physiological and pathological bone turnover. Functional changes in the differentiation and activity of osteoclasts are accompanied by active metabolic reprogramming. However, the biological significance and the in vivo relevance of these events has remained unclear. Here we show that bone resorption of differentiated osteoclasts heavily relies on increased aerobic glycolysis and glycolysis-derived lactate production. While pharmacological inhibition of glycolysis did not affect osteoclast differentiation or viability, it efficiently blocked bone resorption in vitro and in vivo and consequently ameliorated ovariectomy-induced bone loss. Our experiments thus highlight the therapeutic potential of interfering with osteoclast-intrinsic metabolic pathways as possible strategy for the treatment of diseases characterized by accelerated bone loss.

glycolysis and its metabolite lactate were essential for efficient bone resorption. Functional inhibition of glycolysis by 2-Desoxy-D-glucose (2-DG) blocked bone resorption by OCs, which could be rescued by supplementation of either pyruvate or lactate suggesting lactate as an active metabolic intermediate that drives bone resorption. In accordance with a key role of glycolysis during OC-mediated bone resorption in vivo, we observed effective amelioration of ovariectomy-induced bone loss in mice that received 2-DG or a small molecular inhibitor of lactate dehydrogenase A (LDH-A).

Results
Activation of osteoclasts is paralleled by increased aerobic glycolysis. To analyse changes in the metabolic activity of bone resorbing OCs, we isolated murine bone marrow-derived monocytes as OCPs and cultured these cells in M-CSF and RANKL containing medium in the absence and presence of bone powder to simulate a bone resorbing metabolic state in such "activated" OCs (aOCs). The application of bone powder medium did not affect OC fusion capacity, OC number or the expression of OC differentiation marker genes, such as dc-stamp, oscar and cathepsin k (Fig. 1A,B). By performing extracellular flux assays, we compared changes in the extracellular acidification rate (ECAR) as measure of the glycolytic activity between OPCs, nonactivated "resting" OCs (rOCs) and bone resorbing aOCs. OC differentiation resulted in a reduced glycolytic activity in rOCs in comparison to OCPs (Fig. 1C). However, aOCs displayed increased glycolysis, glycolytic reserve (GR) and glycolytic capacity when compared to rOCs, confirming that bone resorption was paralleled by accelerated extracellular acidification and an enhanced aerobic glycolysis (Fig. 1C). Both rOCs and aOCs populations revealed marked elevation of their oxygen consumption rate (OCR), indicated by increased spare respiratory capacity (SRC) and maximal respiration. Moreover, rOCs and aOCs showed an elevated proton leak and basal respiration demonstrating increased mitochondrial respiration during OC differentiation and bone resorption (Fig. 1D).
In addition, we performed metabolic profiling of rOCs and aOCs. In accordance with the data derived from the extracellular flux assays, we observed an increase of metabolites of both glycolysis and the Krebs cycle such as glucose 6-phosphate (G6P) or citrate (Cit), respectively, upon initiation of bone resorption in aOCs (Fig. 1E). Direct measurement of glucose consumption and lactate accumulation in the growth media of rOCs and aOCs revealed that both OC populations substantially consumed glucose. However, lactate production was only significantly elevated in bone-resorbing aOCs, which was in accordance with their enhanced state of aerobic glycolysis (Fig. 1F). When we analysed the metabolic gene expression profile, we found several genes of the glycolytic pathway induced in aOCs (Fig. 1G). In particular, expression of glut1, ldha and pdk4 were increased and provided an explanation for the increase in the rate of glycolysis and enhanced lactate production. On the contrary, ldhb, which catalyses the conversion of lactate and NAD + to pyruvate, was downregulated in aOCs, indicating a metabolic shift towards active aerobic glycolysis and lactate production during bone resorption.
Glycolysis-derived lactate essentially supports bone resorption. Next, we aimed to address the functional consequences of the observed metabolic reprogramming of aOCs including the increase in aerobic glycolysis and lactate production on osteoclast differentiation and bone resorption. We therefore isolated monocytes, assessed OC differentiation and quantified their bone resorbing capacity in the presence of specific inhibitors of oxidative phosphorylation and glycolysis, respectively. Notably, 2dG-mediated inhibition of glycolysis efficiently blocked bone resorption, but did not affect OC differentiation and fusion, whereas rotenone as an inhibitor of mitochondrial complex I, decreased osteoclastogenesis but did only moderately affect resorption activity ( Fig. 2A,B). These results indicated that aerobic glycolysis in OCs was essential for OC activation and bone resorption, but did not affect earlier steps of osteoclastogenesis such as differentiation and fusion events. To determine downstream mediators and metabolites that were responsible for the glycolysis-mediated increase in bone resorption, we assessed the impact of the glycolysis products lactate and pyruvate on the bone resorption capacity of aOCs in the absence and presence of 2dG. While lactate and pyruvate alone did not affect the bone resorbing activity of aOCs, both metabolites rescued the decreased bone resorption caused by inhibition of glycolysis via 2dG (Fig. 2C), which suggested lactate as the active metabolite mediating bone resorption in response to enhanced glycolysis in aOCs.

Inhibition of glycolysis and lactate production ameliorate ovariectomy-induced bone loss.
To assess the potential of OC-mediated glycolysis as therapeutic target for diseases characterized by a pathologi-  www.nature.com/scientificreports/ cally enhanced bone resorption, we decided to study the effect of blocking glycolysis and lactate production during a mouse model of ovariectomy-induced bone loss. Therefore, we treated mice that displayed an established osteoporosis (treatment start 2 weeks post ovariectomy) with the glycolysis inhibitor 2dG for 6 additional weeks before bone structure was analysed by μCT (Fig. 3A). 2dG treatment was well tolerated. As expected, mice that had undergone ovariectomy displayed an increased weight gain over the study period of 8 weeks, which was slightly decreased by treatment with 2dG ( Fig. 3B). Analysis of the trabecular microstructure of the spine showed that 2dG slightly decreased bone density in the sham-operated group, whereas we observed an increase in bone density upon 2dG treatment in mice that had undergone ovariectomy (Fig. 3C,D). 2dG treatment resulted in an increased bone volume/total volume (BV/TV), an increased bone mineral density (BMD) www.nature.com/scientificreports/ and an increased trabecular thickness in ovariectomized mice, which was in accordance with a block of pathologic bone resorption in these mice. We additionally validated these results and our in vitro findings in a second round of experiments where we treated mice with the LDHA inhibitor GSK2837808A to selectively interfere with lactate production in vivo. GSK2837808A inhibited the bone resorbing activity of osteoclasts to a similar extend as 2dG without cytotoxic effects in vitro (Suppl. Fig. 1A,B). Treatment was started 2 weeks after ovariectomy in mice that already had developed bone loss (Fig. 3E). In accordance with our previous results, block of lactate synthesis resulted in a slight decrease in bone density in sham-operated mice, whereas GSK2837808A treatment resulted in a significant improvement of ovariectomy-induced bone loss (Fig. 3F,G). Here, we observed an increased BV/TV, an increased trabecular thickness as well as an increased BMD in ovariectomized mice that had received GSK2837808A.

Discussion
Increasing data highlight the mutual influence of cellular metabolic processes and the functional properties of innate and adaptive immune cells. Among others, macrophages were shown to undergo a defined metabolic reprogramming in response to different activation stimuli. Pro-inflammatory classically-activated macrophages e.g. increase their glycolytic activity and shut down their mitochondrial respiration, whereas anti-inflammatory alternatively-activated macrophages show an increase in mitochondrial respiration. These contrasting metabolic adaptions of individual macrophage subsets seem to support distinct activation and differentiation programs, thereby promoting either onset or resolution of inflammation 8 . Our current data confirm that osteoclast, as mononuclear phagocyte-derived cells that settle the bone microenvironment, undergo specific metabolic adaptions during differentiation and bone resorption as well.
Notably, and in contrast to many other immune cells, bone resorbing and activated osteoclasts are not characterized by an isolated, but by a simultaneous increase of both glycolytic activity and mitochondrial respiration when compared to resting osteoclasts. This observation suggests that the process of bone resorption substantially increases the metabolic and energetic requirements of osteoclasts. This adaptation might be merely linked to an increased demand in energy. Alternatively, it points to the necessity of a provision of specific and essential metabolites that derive from glycolysis and/or mitochondrial respiration and support the functional properties of osteoclasts during bone resorption. Our data primarily support the latter scenario and suggest that glycolysisderived lactate is essential for the efficient resorption of bone by osteoclasts. Block of oxidative phosphorylation, in turn, only moderately affected bone resorption, although osteoclast differentiation was clearly impaired. Block of glycolysis by 2dG or block of the conversion of pyruvate to lactate by the LDHA-inhibitor GSK2837808A accordingly interfered with regular bone resorption, which could be rescued by the restoration of regular lactate levels. The exact role of lactate during this process remains unclear at the moment. Our in vitro experiments show a substantial increase in extracellular lactate upon initiation of bone resorption. A likely possibility is therefore a role of lactate during the acidification of the osteoclast resorption pit, which is an essential step during bone resorption by osteoclasts, facilitates the activation of acid proteases that brake down bone matrix, and additionally dissolves bone minerals.
Our data additionally show that blocking glycolysis and lactate production represents efficient strategies to interfere with pathological osteoclast-mediated bone loss during ovariectomy as a model of postmenopausal osteoporosis that is usually characterized by increased differentiation and activation of osteoclasts 9 . Although such a strategy significantly increased bone mass in osteoporotic mice, healthy mice displayed a slightly decreased bone density upon block of glycolysis. Although the underlying reasons remain to be elucidated, block of glycolysis likely affects other cell types such as osteoblasts and osteocytes that are equally relevant for a regular bone homeostasis. Especially osteoblast are dependent on glycolysis to form new bone 10 , although they equally require mitochondrial respiration and fatty acids to mineralize newly formed extracellular matrix 11 .
Taken together, our data identify aerobic glycolysis and osteoclast-mediated lactate production as essential steps during bone resorption and suggest that blocking glycolysis and lactate production in osteoclasts represents a potential therapeutic strategy during diseases that are characterised by accelerated osteoclast-mediated bone loss such as postmenopausal osteoporosis and rheumatoid arthritis.

Methods
The authors confirm that all methods were carried out in accordance with relevant guidelines and regulations. The ethical approval for all animal experiments was carried out in accordance with relevant guidelines and regulations by the ethical committee of the Friedrich Alexander University of Erlangen-Nuremberg (FAU), Germany.

Mice.
Mice were maintained at the specific pathogen-free animal care facility (FPZ) of the University of Erlangen-Nuremberg and housed in a room at 23 ± 2 °C, with 50 ± 10% humidity and a 12-h light/dark cycle (lights on from 08:00 a.m. to 08:00 p.m.). All mice were allowed free access to water and regular rodent chow.

Ovariectomy-inducd osteoporosis (OVX).
Female C57BL/6JRj wild-type mice were obtained from Janvier Labs and experiments were performed according to guidelines of laboratory animal care and use. All efforts were made to reduce the number of animals tested and their suffering. For ovariectomy, mice (12 weeks of age) were anaesthetized with a ketamine-xylazine (KX) solution and bilaterally ovariectomized, while ovaries of the sham group were left intact. The ovariectomized mice were allowed to recover for two weeks to ensure the development of osteoporosis. After 6 full weeks of treatment with vehicle, 2d or GSK 2837808A, all animals were killed via cervical dislocation under CO 2 anaesthesia and one tibia was excised for histological analyses and one for microcomputer tomography (μCT) imaging. Successful ovariectomy was examined at the day of preparation by anatomical analysis of the ovaries. Cell culture. Bone marrow derived monocytes (BMDMs) were isolated as previously described and differentiated into osteoclast (OCs) 8 . Briefly, hematopoietic bone marrow cells were purified from tibial bone with a 70-μm cell strainer, cultured overnight and stimulated with appropriate growth medium (MEM Alpha Medium 1 × GlutaMAX, GIBCO; #32571-028, containing 10% L929 conditioned medium, 10% FCS and 1% penicillin/streptomycin) at 37 °C with 5% CO 2 . OCPs were cultured in OC-growth medium (MEM Alpha Medium 1 × GlutaMAX, GIBCO; #32571-028, containing 10% L929 conditioned medium, 10 ng/ml RANKL, 10% FCS and 1% penicillin/streptomycin) for 24 h. Osteoclast differentiation was quantified by counting multinucleated (> 3 nuclei) TRAP + cells on day 4 of OC culture using the Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma-Aldrich, # 387A). Osteoclast fusion events were quantified by counting the nuclei per osteoclast.  www.nature.com/scientificreports/ RT-PCR analysis. Total RNA was isolated from cells using peqGOLD TRIFast (peqlab, Germany). 1 µg was used for the first-strand complementary DNA synthesis (Amersham Biosciences), which was then used for SYBR Green-based quantitative RT-PCR as described previously 12  growth medium was replaced by Seahorse XF Base Medium and supplemented with glucose (10 mM), sodium-pyruvate (200 mM) and glutamax (200 mM). Further, medium was warmed up to 37 °C and pH was subsequently adjusted to 7.4 ± 0.1. For Glyco Stress assay, no glucose and pyruvat was added to Seahorse XF Basismedium. Following incubation in an incubator without CO 2 at 37 °C for 60 min, basal OCR and ECAR were recorded for 105 min. Mito Stress assay was performed by sequential addition of 20 µM oligomycin (inhibitor of ATP synthesis), 10 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, uncoupling agent) and 10 μM rotenone/antimycin A (inhibitors of complex I and complex III of the respiratory chain, respectively). Glyco Stress assay was performed by sequential addition of 100 mM glucose, 20 µM oligomycin and 500 mM 2dG. Parameters such as ATP-linked OCR, maximal OCR, spare respiratory capacity (SRC) and glycolysis were evaluated using the Wave XFe Analyzer software Wave Desktop 2.6 (PC only) and Wave Controller 2.6 (https ://www.agile nt.com/). Glucose and lactate measurements. Glucose and lactate content were determined as mg/dl in cell culture supernatants using a SuperGLcompact (Hitado, Möhnesee, Germany) according to the manufacturers standard operating procedure.

Measurement of glycolysis and Krebs cycle metabolites. Phosphorylated intermediates and car-
boxylates were extracted with perchloric acid from xx to xx mg samples of shock-frozen xxx tissue for four biological replicates as described previously 13 applying ionchromatography with an ICS3000 HPLC-system (Dionex) and ESI/MS/MS detection using a QTrap3200 Triple-Quadrupole massspectrometer with turbo V ion source (Applied Biosystems) operated in multiple reaction monitoring mode. Cytotoxicity assay. Cytotoxicity assay was performed with CytoTox 96 Non-Radioactive Cytotoxicity Assay from Promega as a measurement of lactate dehydrogenase (LDH) release into the supernatant according to the manufactors protocol. During experimental treatment via bone resorption assay, supernatant samples were cleared from non adherent cells and transferred to a 96 plate and an equal volume of CytoTox 96 reagent is added to each well and incubated for 30 min. Stop solution is added and the absorbance signal is measured at 490 nm in a plate reader. Results were calculated by subtracting the average values of the culture medium background from all values of experimental wells to compute percent cytotoxicity. Statistical analysis. Statistical analyses were performed using GraphPad Prism 8 software (https :// www.graph pad.com/scien tific -softw are/prism /). Results are depicted as median ± interquartile range (IQR) if not stated otherwise. For a two-group comparison, a Student's t-test was applied if the pretest for normality (D' Agostino-Pearson normality test) was not rejected at the 0.05 significance level; otherwise, a Mann-Whitney U-test for nonparametric data was used. P values less than 0.05 were considered significant. Results are expressed as mean ± SE (SEM). No statistical method was used to predetermine sample size.