Targeting Bone Marrow Lipid Metabolism with Adiponectin Receptor Agonist for Diabetic and Postmenopausal Osteoporosis

doi:10.21203/rs.3.rs-3334811/v1

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Targeting Bone Marrow Lipid Metabolism with Adiponectin Receptor Agonist for Diabetic and Postmenopausal Osteoporosis

https://doi.org/10.21203/rs.3.rs-3334811/v1

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Osteoporosis is a malady of multiple cell types associated with the bone marrow (BM) fat accumulation. This study focused on understanding the role of adiponectin in osteoblast and osteoclast lipid metabolism in diabetic and ovariectomized mice with osteoporosis and exploring the effects of the adiponectin receptor (AdipoR) agonist, AdipoRon. AdopoRon prevented systemic bone resorption and oxidative stress, and excessive lipid accumulation in BM. AdipoRon activated AMPK and PPARɑ through AdipoR1 and AdipoR2, leading to a reduction in lipotoxicity-induced osteocyte apoptosis, oxidative stress, and inflammation with M1 polarization. All these changes increased osteogenic RUNX2/OPG/FOXO1 expression, decreased osteolytic RANKL/PPARγ expression, and preserved bony phenotypes and growth plate thicknesses in the lumbar vertebrae and femur. AdipoR1/R2 knockout in pre-osteoblasts and osteoclasts in high-glucose and palmitate media confirmed the positive action of AdipoRon on bone formation, which was achieved by activating AMPK and PPARɑ to the same degree through both AdipoR1 and AdipoR2. These activations were closely associated with the amelioration of osteocyte apoptosis, oxidative stress, inflammation, and autophagy induced by high-glucose and palmitate, which were controlled by regulating lipid metabolism. We revealed that AdipoRon may safeguard lipotoxicity in BM in diabetic and ovariectomized osteoporosis by modulating lipid metabolism in osteoclasts and osteoblasts.

Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis

Health sciences/Diseases/Endocrine system and metabolic diseases/Diabetes/Type 2 diabetes

Adiponectin

Diabetic osteoporosis

Lipotoxicity

Postmenopausal osteoporosis

The crosstalk between bone remodeling and energy metabolism is a paramount determinant of bone phenotype. Bone remodeling plays a crucial role in the longitudinal growth and repair of bone micro- and macro-damages; its disruption leads to osteoporosis ¹. Under homeostatic conditions, a balance exists between osteogenesis and adipogenesis, in which bone marrow adipocytes support local energy utilization and secrete paracrine factors to stimulate osteoblast activity, favoring bone mass formation ². In adult humans, bone marrow (BM) adipose tissue comprises 50–70% of the BM cavity. However, during diabetes and gonadal failure, this bone-fat balance is disrupted, leading to adipose tissue accumulation in the BM and low bone turnover, thus contributing to osteoporosis ^3–5. BM adiposity, characterized by the expansion of adipocyte size but not density, has been observed in populations with obesity, diabetes, or elderly persons. There is a strong correlation between BM adiposity and bone loss such that it is associated with a markedly increased risk of hip fractures ^3,6.

Although the relationship between BM adiposity and osteopenia is unclear, fat accumulation in the BM interferes with bone formation. BM mesenchymal stem cells primarily differentiate into osteoblasts and adipocytes within the skeleton. If this differentiation is imbalanced with an increase in adipocytes and a decrease in osteoblasts, osteoporosis can occur ⁷. BM adipocytes secrete adipokines, which lead to the increased secretion of cytokines, such as tumor necrosis factor α (TNF-α), interleukin (IL)-1, IL-6, and receptor activator of the NF-kB ligand (RANKL). Adipokines and cytokines promote osteoclast differentiation and simultaneously inhibit osteoblast formation ⁸. With aging, excessive osteocyte apoptosis is correlated with increased oxidative stress, favoring osteoclastogenesis and hence a disproportionate increase in bone remodeling and loss ⁹.

Adiponectin, an adipokine secreted mainly by adipocytes, plays a crucial role in regulating energy homeostasis and glucose and fatty acid metabolism. Adiponectin receptor (AdipoR) 1 and 2 are integral membrane proteins that mediate adiponectin's metabolic actions ¹⁰. These proteins are derived from the mesenchymal stem cell lineage and are found in precursors, such as pre-osteoblasts, pre-osteoclasts, and chondrocytes, suggesting that AdipoR signaling may be involved in bone and cartilage metabolism ¹¹. Although most in vitro studies have confirmed that adiponectin promotes osteoblastogenesis ^12–15 and inhibits osteoclastogenesis by modulating RANKL ¹⁶, its potential to stimulate the cellular plasticity of osteoblasts toward adipocytes and its further association with decreased bone density cannot be overlooked ^17–20. Furthermore, although mixed results have been observed for adiponectin, increased bone marrow adipogenesis can adversely affect bone remodeling through a direct negative effect on mesenchymal stem cell osteoblast differentiation. Recent studies investigating the impact of pharmacological activation of AdipoRs by the synthetic agonist AdipoRon revealed an inhibition of mature osteoclast activity through AMP-activated protein kinase (AMPK), a master switch of energy homeostasis ^21,22. Therefore, this has drawn our attention to the potential role of AdipoR agonism in promoting bone remodeling by modulating adipogenesis.

Recently, there has been extensive research into lipid metabolic alterations in various bone diseases, including osteoporosis. These alterations can affect the function and phenotype of osteoblasts and osteoclasts ²³, which primarily rely on glycolysis and oxidative phosphorylation for their essential energy needs. Additionally, both osteoblasts and osteoclasts can derive an incidental cell energy source from de novo lipogenesis. Therefore, any disruption to the lipid metabolism process can impair osteoblast function ²⁴. For example, dyslipidemia causes an increased lipid load and stimulates the inflammatory response in osteoclasts, ultimately resulting in osteoporosis ²⁵. Despite this understanding, the efficacy of lipid metabolism-targeting drugs for bone loss has not yet been fully evaluated ²⁵.

In this study, we examined AdipoR1/R2 expression in bone tissue after AdipoRon application. We hypothesized that AdipoRon can ameliorate osteoporosis in the lumbar vertebrae and femurs of diabetic and ovariectomized mice. This is achieved by increasing the expression of both AdipoR1/R2 and activating their target molecules via the AMPK/peroxisome proliferation-activated receptor (PPAR)α pathway, as well as associated downstream pathways that regulate lipid metabolism in the bone, especially in osteoblasts and osteoclasts.

AdipoRon treatment increased bone volume and density and decreased fat volume in the bone marrow of the femurs and vertebrae of db/db mice.

The body weight, blood glucose, HbA1c, as well as the serum levels of total cholesterol, triglycerides, and non-esterified fatty acid and urinary levels of 8-hydroxy-deoxyguanosine (8-OH-dG) and 8-isoprostane, were significantly higher in the db/db mice groups than in the db/m mice groups (Suppl. Table 1). In contrast, the serum adiponectin levels were significantly lower in db/db mice groups, and this trend was maintained after AdipoRon treatment. After 4 weeks of AdipoRon treatment in db/db mice, there were no changes in body weight, blood glucose, or HbA1c, suggesting that AdipoRon had no direct effects on glucose regulation. However, serum triglycerides and urinary 8-OH-dG and 8-isoprostane were reduced by 26% and 65%, respectively, indicating that AdipoRon improved systemic hypertriglycemia and oxidative stress in db/db mice groups. The db/db mice showed characteristic features of osteoporosis, including increased serum tartrate-resistant acid phosphatase (TRAP/ACP5) and urinary deoxypyridinoline (DPP), as well as decreased serum osteocalcin, compared to the db/m group. However, these effects were reversed by AdipoRon treatment (Suppl. Figure 2). In the diabetic mice, bone volume increased, and the bone surface-to-volume ratio decreased in both the femurs and vertebrae after AdipoRon treatment. There was an increase in trabecular bone number and thickness, and a decrease in its separation, resulting in an increase in bone mineral density (Fig. 1a, 3a). Microscopic and gross examinations of the bone marrow in diabetic mice also revealed an increase in bone volume and bone mineral density, as well as a decrease in bone fat volume (Fig. 1a, 3a). Furthermore, AdipoRon treatment increased the thickness and number of chondrocytes in the growth plate and decreased the fat volume (Fig. 1b). None of these bone-related changes were noted in the db/m mice groups after AdipoRon treatment.

AdipoRon treatment activated AdipoR1/R2 and their downstream pathways, leading to enhanced lipid metabolism. This decreased inflammation, apoptosis, and oxidative stress, resulting in increased osteoblastogenesis and decreased osteoclastogenesis in the femurs and vertebrae of db/db mice

AdipoR1 and AdipoR2 expression in the BM increased after AdipoRon treatment, followed by an increase in downstream signaling pathways involving pAMPK, PPARα, and PPARγ coactivator (PGC)-1α, and a decrease in pFOXO1 expression (Fig. 2a, b, 3b, c). Phosphorylated acetyl-CoA carboxylase (pACC) expression increased, while perilipin 1 and 2, sterol regulatory element-binding protein (SREBP), carbohydrate response element-binding protein (ChREBP), and stearoyl-CoA desaturase (SCD)-1 expression decreased, indicating inhibited de novo lipid production and enhanced lipid metabolism following AdipoRon treatment (Fig. 2c, 3d). Regarding markers for bone remodeling and reabsorption, RANKL and PPARγ expression decreased, while OPG and RUNX2 expression increased, suggesting the activation of osteoblastogenesis and the inhibition of osteoclastogenesis after AdipoRon treatment (Fig. 2d, e, 3e, f). AdipoRon treatment reduced the expression of inflammatory, autophagic, apoptotic, and oxidative stress markers, leading to an M1-like to M2-like shift in macrophage polarization in diabetic mice. AdipoRon treatment increased SOD1, SOD2, NQO1, HO-1, and LC3-II expression and decreased NOX4, TNF-α, TUNEL, cytoplasmic and nuclear NF-kB, CD68, ArgII, and iNOD expression (Fig. 2f-h, 3g-i).

AdipoRon treatment led to an increase in bone volume and density, and a decrease in fat volume in the bone marrow of femurs and vertebrae of ovariectomized mice.

In ovariectomized mice, similar to db/db mice, we observed increased urinary DPP and decreased serum osteocalcin levels compared to non-ovariectomized control mice. However, these changes were reversed by the administration of AdipoRon, regardless of the dosage (Suppl. Figure 4). Based on the changes in these markers, AdipoRon treatment appears to shift the balance from bone resorption to bone formation. Furthermore, in both the femurs and vertebrae of the ovariectomized mice, bone volume increased in a dose-dependent manner, whereas the bone surface-to-volume ratio decreased with AdipoRon treatment. Their trabecular bone number and thickness increased, while their trabecular separation increased, resulting in an increase in bone mineral density. Microscopic and gross examinations (transaxial and sagittal sections) of the bone marrow revealed a discernible increase in bone volume and bone mineral density, and a decrease in fat volume (Fig. 4b, 5a). AdipoRon treatment increased the thickness and number of chondrocytes in the growth plate and decreased fat volume in the ovariectomized mice group (Fig. 4a).

AdipoRon treatment activated AdipoR1/R2 and their downstream pathways, leading to enhanced lipid metabolism. This in turn decreased inflammation, apoptosis, and oxidative stress, resulting in increased osteoblastogenesis and decreased osteoclastogenesis in the femurs and vertebrae of ovariectomized mice

After AdipoRon treatment, AdipoR1 and AdipoR2 expression increased regardless of the dosage. Subsequently, downstream signaling pathways involving AMPK, PPARα, and PGC-1α increased (Fig. 4c, 5b). Investigation of the target molecules involved in lipid metabolism revealed an increase in pACC expression and a decrease in perilipin 1 and 2, SREBP, ChREBP, and SCD-1 expression, indicating enhanced lipid metabolism after AdipoRon treatment (Fig. 4d, 5c). Markers for bone remodeling and reabsorption showed decreased RANKL and PPARγ expression, as well as increased OPG and RUNX2 expression, suggesting the activation of osteoblastogenesis and inhibition of osteoclastogenesis after AdipoRon treatment (Fig. 4c, 5b). AdipoRon treatment reduced the expression of inflammatory, autophagic, apoptotic, and oxidative stress markers. Specifically, LC3II (Fig. 4c, 5b),TNF-α, TUNEL,CD68, ArgII, and iNOS expression decreased (Fig. 4e, f, 5d, e).

AdipoRon treatment modulated the expression of target molecules involved in lipid and bone metabolism, leading to enhanced osteoblastogenesis in cells cultured with high-glucose (HG) and palmitate acid (PA) media.

Pre-osteoblasts (MC3T3-E1 cells) cultured in both HG and PA media displayed a reduction in the Alizarin red-positive area and density, which was ameliorated by AdipoRon treatment (Fig. 6a). Furthermore, AdipoRon increased pAMPK, PPARα, PGC-1α, pACC, and LC3II expression and decreased pFOXO1, SREBP-1c, ChREBP, SCD-1, and perilipin 1 and 2 expression (Fig. 6b, c). AdipoRon also increased RUNX2 and OPG expressions, and decreased PPARγ and RANKL expressions (Fig. 6b). To determine which AdipoR is responsible for activating downstream effectors of the pathway, cells cultured in HG and PA media were transfected with small interfering RNAs (siRNAs), thereby silencing the genes encoding AdipoR1 and AdipoR2 (Fig. 6d, e). Western blotting studies of osteoblasts transfected with either AdipoR1 or AdipoR2 siRNA revealed that the decreased expression of pAMPK, PPARα, PGC-1α, RUNX2, OPG, and pACC increased and the increased expression of pFOXO1, PPARγ, RANKL, perilipin 1 and 2, SREBP-1c, ChREBP, and SCD-1 decreased after AdipoRon treatment, confirming that the expression of these downstream target molecules is AdipoR-dependent (Fig. 6d, e). Interestingly, these changes were achieved by activating the same degree of AMPK and PPARɑ through each of AdipoR1 and AdipoR2. Consequently, it appears that both AdiopR1 and AdiopoR2 are essential for achieving full AdipoRon-induced AMPK and PPARɑ activation. Furthermore, we found that HG did not potentiate PA-induced lipotoxicity in osteoblasts, indicating that HG and PA had no synergistic effect on osteoblasts.

AdipoRon modulated the expression of target molecules involved in lipid and bone metabolism, leading to reduced osteoclastogenesis in cells cultured with HG + PA media and treated with RANKL.

Osteoclasts (RAW264.7 cells) cultured in HG and PA media were first treated with RANKL alone and then with AdipoRon. The number of multinucleated osteoclasts increased with RANKL treatment but decreased with AdipoRon treatment, indicating a reduction in osteoclastic activity conferred by AdipoRon (Fig. 7a, b). AdipoRon increased pAMPK, PGC-1α, PPARα, pACC, and LC3II expression, and decreased pFOXO1, perilipin 1 and 2, SREBP, ChREBP, and SCD-1 expression in cells treated with RANKL (Fig. 7c, d). AdipoRon treatment increased RUNX2, RANKL, and OPG expression, and decreased PPARγ expression in cells treated with RANKL (Fig. 7c). Furthermore, AdipoRon treatment attenuated the increase in dihydroethidium (DHE)- and TUNEL-positive cells and restored the expression of LC3II in cells cultured in HG and PA media and treated with RANKL (Fig. 7e-h). Next, we used osteoblasts transfected with either AdipoR1 or AdipoR2 siRNA to confirm the effect of AdipoRon (Fig. 7i, j). Western blotting studies of osteoblasts transfected with either AdipoR1 or AdipoR2 siRNA revealed that the decreased pAMPK, PPARα, PGC-1α, RUNX2, OPG, and pACC expression increased, and the increased pFOXO1, PPARγ, RANKL, perilipin 2, SREBP-1c, and ChREBP expression decreased after AdipoRon treatment, confirming that the expression of these downstream target molecules are AdipoR-dependent (Fig. 7i, j). Similar to the observations in osteoblasts, these changes were also achieved by activating the same degree of AMPK and PPARɑ through each of AdipoR1 and AdipoR2. In addition, PA-induced lipotoxicity was equally noticeable in both LG and HG conditions, indicating that it was not affected by glucose concentration.

In our study, we demonstrated that osteoporotic bone loss in male diabetic and female ovariectomized mice was characterized by an increase in BM adipogenesis and reduced bone formation. AdipoRon treatment prevented bone loss by reducing BM adiposity and enhancing lipid metabolism. The primary effect of AdipoRon treatment was altered lipid metabolism, resulting in decreased lipid synthesis and increased fatty acid oxidation, as reflected by the activation of target molecules, both AMPK and PPARα. We confirmed this process by using AdipoR1 and AdipoR2 siRNAs in osteoblastic MC3T3-E1 and osteoclastic RAW264.7 cells cultured in HG and PA media. AdipoRon treatment increased pACC expression and decreased perilipin 1 and 2, ChREBP, SREBP-1c, and SCD-1 expression. Enhanced lipid metabolism in osteoblasts and osteoclasts by AdipoRon was associated with amelioration of oxidative stress, inflammation, autophagy, and apoptotic markers in mouse femurs and vertebrae.

AdipoRon treatment inhibited apoptotic cell death and promoted autophagy in the BM y activating AMPK. We presumed that reduced FOXO1 phosphorylation after AdipoRon treatment played a crucial role in preventing osteoporosis in these mouse models. FOXO1 is a key modulator of redox signaling and bone homeostasis. When activated by reactive oxygen species, it translocates to the nucleus, where it upregulates the activities of the antioxidant enzyme, SOD, and mediates cell antioxidant stress, apoptosis, and autophagy ²². Perilipin 1 and 2 act as physical barriers to protect lipid droplets, which mainly contain triacylglycerols, from lipase action and inhibit autophagic lipolysis ²⁶. However, recent studies have shown that bone marrow adipocyte lipolysis is necessary for bone homeostasis and trabecular and cortical bone regeneration under stress conditions ²⁷. The inhibitory effect of AdipoRon on perilipin 1 and 2 expression in osteoblasts, osteoclasts, and the BM might be associated with enhancement of the osteogenic response related to increased autophagic lipolytic activity. Consistent with this finding, a recent study found that the levels of the lipogenesis protein perilipins correlated with higher lipid accumulation in osteoporotic BM ²⁸. Furthermore, TNF-α in the marrow stimulates chondrocyte apoptosis and accelerates bone loss via FOXO1, especially in diabetes ^27,29. AdipoRon treatment improved the viability of osteocytes and chondrocytes in the femur and maintained their growth plate thickness by inhibiting FOXO1 phosphorylation-induced bone cell apoptosis. This led to subsequent antioxidative and anti-inflammatory effects conferred by Nrf2 activation and NF-κB/TNF-α inhibition, respectively.

Adipocytes and osteoblasts originate from the same mesenchymal progenitor, and their differentiation into either lineage is influenced by various signals ^30–33. PPARγ, a key adipogenic transcription factor, can negatively affect skeletal homeostasis by decreasing bone formation via suppression of osteoblastogenesis and by increasing bone resorption via stimulation of osteoclastogenesis ³⁴. In contrast, AMPK plays a vital role in osteoblast differentiation and mineralization, as indicated by the increased expression of RANKL and the RANKL/OPG ratio in primarily cultured osteoblasts from AMPK-deficient mice ³⁵. In this study, AdipoRon, with its pro-osteoblastogenic and anti-osteoclastogenic activities, enhanced lipid metabolism by activating the adiponectin receptor-mediated upregulation of AMPK/PPARα and downregulating the PPARγ pathways. AdipoRon promoted Runx-2 expression and the OPG/RANKL ratio in the lumbar and femoral bones of both mouse models and during the early stages of osteoblast differentiation in osteoblastic MC3T3-E1 and osteoclastic RAW264.7 cells cultured in HG and PA media. Adiponectin deficiency favors the differentiation of osteoclast precursors into M1-like macrophages ³⁶. However, our results demonstrated that AdipoRon treatment led to a shift from M1-like to M2-like macrophage polarization, thereby reducing the concentration of M1 macrophages in relation to increased BM adiposity. Our findings suggest that AdipoRon exhibits anti-osteoclastogenic activity by upregulating the OPG/RANKL ratio.

A previous study using human osteoblasts showed that adiponectin treatment increased RANKL expression and suppressed OPG expressions ²⁰. However, transgenic mice overexpressing human full-length adiponectin did not show significant differences in RANKL and OPG expression when compared to wild-type mice ³⁷. These conflicting findings regarding the effects of adiponectin and AdipoRon on the OPG/RANKL ratio may be partially attributed to differences in the experimental models and cell culture media. Moreover, regarding the association between adiponectin and reduced bone mass, circulating adiponectin, particularly in its full-length form rather than its globular form, indirectly inhibits bone mass by increasing insulin sensitivity and inhibiting insulin action in tissues ^18,38. In contrast, previous studies have shown that AdipoRon activates adiponectin receptors and their downstream signaling pathways without affecting the circulating adiponectin level ³⁹. Thus, AdipoRon may exert favorable effects by bypassing the limitations associated with chronic adiponectin exposure. However, it is important to note that many of the beneficial effects conferred by this adiponectin mimetic may require chronic exposure to be fully manifested.

Interestingly, AdipoRon treatment improved femoral growth plate thickness and lumbar and femoral osteoporosis in both mouse models. Previous studies have shown that adiponectin directly stimulates chondrocyte proliferation, proteoglycan synthesis, matrix mineralization, collagen X, and RUNX2 expression in the ATDC5 cell line, which is a widely used in vitro model of chondrogenesis. The expression of these characteristic markers of late-phase chondrogenic differentiation was upregulated, suggesting that adiponectin is a potent stimulator of chondrocyte maturation ⁴⁰. This finding is consistent with the fact that, similar to adiponectin, AdipoRon may directly influence chondrocytes by promoting proliferation and maturation through AdipoR1/R2 activation, favoring bone elongation. Overall, AdipoRon appears to promote pre-osteoblast differentiation and mineralization capacity. However, it has a significant inhibitory effect on the differentiation of murine macrophage precursors into mature osteoclasts in vitro, especially in the presence of RANKL. In the liver, targeted disruption of AdipoR1 led to the loss of adiponectin-induced AMPK activation, while disruption of AdipoR2 resulted in decreased activity of PPARɑ signaling pathway ⁴¹. Interestingly, in our current study, AdipoRon activated both AMPK and PPARɑ to the same degree through both AdipoR1 and AdipoR2 in the bone, which was in contrast to the liver. Further research is required to explore this phenomenon. We speculate that this shared signaling pathway might serve as a safeguard to prevent osteoporosis in the event of a deficiency in one receptor.

Contrary to findings in other cells ^42,43, we did not observe a synergistic and augmented effect of HG on PA-induced lipotoxicity in osteoblasts and osteoclasts. In these cells, the metabolism of glucose and free fatty acids is intertwined, and glucose is the primary source of energy, producing adenosine triphosphate ⁴⁴. It is worth noting that fatty acids derived from dietary fat or from triglyceride hydrolysis in fat cells are an effective substrate for energy production. Oxidation of the same weight of carbohydrate or protein produces less than half the amount of energy produced by the oxidation of fatty acids ⁴⁵. To this day, there is limited understanding of the detrimental effects of HG combined with dyslipidemia on bone mass and the processes of bone formation and resorption. Typically, the effects of lipotoxicity are not organ specific and instead affect the fundamental cellular processes occurring in most cells ¹⁴. Further studies are required to elucidate the relationship between HG and lipid metabolism in the maintenance of bone density and phenotype in response to diabetes and postmenopausal osteoporosis. In the present study, there was no synergistic lipotoxic interaction between HG and PA in osteoblasts and osteoclasts, indicating that HG or PA alone could cause sufficient osteoporoic phenotypes.

We acknowledge certain limitations of the present study. In this investigation, we aimed to explore the potential of AdipoRon, an adiponectin receptor agonist, to protect against lipotoxicity in the BM. Our study utilized mice models to examine the modulation of fat–bone imbalance in both diabetic and ovariectomized osteoporosis. However, it is important to note that the use of mouse models may not accurately reflect real-life clinical scenarios. Consequently, future studies should prioritize investigating whether adiponectin receptor agonists exhibit a protective effect in human cases of diabetic and postmenopausal osteoporosis.

In conclusion, AdipoRon prevented osteoporotic changes in diabetic and ovariectomized mice models by activating both AdipoR1/R2 and their respective target molecules, phospho-AMPK/PPARα, and the associated downstream lipid metabolism signaling pathways in osteoblasts and osteolclasts in the BM. These lipid metabolic effects confer benefits in terms of apoptosis, autophagy, oxidative stress, and inflammation in BM tissue and cells. Our findings provide insight into the influence of adipogenic events on osteoclast differentiation and the intracellular mechanisms that ultimately promote osteoblastogenesis (Fig. 8). Targeting these pathways could potentially promote bone regeneration in bone loss disorders, making AdipoRon, an adiponectin receptor agonist, a promising therapeutic option.

Experiment 1

Six-week-old male C57BLKS/J db/m and db/db mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). The mice were randomly divided into four groups of eight mice each: db/m control, db/m with AdipoRon treatment (db/m + AdipoR 30 mg/kg), db/db control, db/db with AdipoRon treatment (db/m + AdipoR 30 mg/kg). AdipoRon (30 mg/kg; Sigma-Aldrich; No. SML0998) was mixed with a standard chow diet and provided to mice at 16 weeks old for 4 weeks. At week 20, all animals were anesthetized using 30 mg/kg tiletamine plus zolazepam (Zoletil; Virbac, Carros, France) and 10 mg/kg xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany). The effects of AdipoRon treatment were compared between the groups (Suppl. Figure 1).

Experiment 2

To examine the effect of AdipoRon on estrogen deficiency-induced bone loss, 7-week-old female C57BL/6 sham-operated (Sham) and C57BL/6 ovariectomized (OVX) mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). The mice were randomly divided into six groups of eight mice each: sham-operated control (Sham), Sham with AdipoRon treatment (OVX + AdipoR 2.5 mg/kg), Sham with AdipoRon treatment (OVX + AdipoR 25 mg/kg), OVX-operated control, OVX with AdipoRon treatment (OVX + AdipoR 2.5 mg/kg), and OVX with AdipoRon treatment (OVX + AdipoR 25 mg/kg). The Sham mice were incised and sutured, and ovariectomy was not performed. The mice in the OVX and OVX + AdipoRon groups underwent bilateral ovariectomy. After 7 postoperative weeks, AdipoRon (2.5 mg/kg and 25 mg/kg; Sigma, No. SML0998) was mixed with a standard or regular chow diet (control) for 6 weeks, after which all the mice were sacrificed (Suppl. Figure 3).

Biochemical measurements

Blood was collected at the time of harvest, allowed to clot at room temperature for 30 min, and centrifuged at 3,000 rpm for 10 min. Serum TRAP/ACP5 and osteocalcin were measured using Mouse TRAP and OC assay kits (LSBIO, No. LS-F28187; MyBioSource, No. MBS725134) according to the manufacturer’s protocol. Urine levels of 8-OH-dG (Oxis, No. 21026, 21048), a marker of oxidative DNA and lipid damage, and DPP, a marker of bone resorption, were measured using commercial enzyme-linked immunosorbent assay kits (MyBioSource, No. MBS760647) according to the manufacturer's protocol.

Microcomputed tomography

Computed tomography (CT) of the femur and fifth lumbar vertebrae was performed on the experimental mice. The images were reconstructed into a three-dimensional structure using micro-CT (SkyScan 1172; Bruker) with a voxel size of 5.9 mm. The X-ray tube voltage and current were 60 kV and 167 mA, respectively. The exposure time was 440 ms. X-ray projections were obtained at 0.4° intervals with a scanning angular rotation of 360°. Our analysis variables were bone mineral density, bone volume/total volume, percent bone volume, bone surface/bone volume, bone-specific surface (in mm^− 1), trabecular thickness (in mm), trabecular separation (in mm), and trabecular number (in mm^− 1). The bone microstructure was assessed according to the guidelines provided by Bouxsein et al ⁴⁶.

Decalcification

The femur and fifth lumbar vertebrae were prefixed in 4% paraformaldehyde for 24 h. Samples were then rinsed in phosphate buffered saline (PBS) for 24 h and incubated with 10% EDTA (pH 7.4). Decalcification was performed at 4°C under continuous shaking. The decalcifying solutions were changed daily. The total decalcification time was 14 days. The decalcification process ended when the bone was easily penetrated by a needle without any force. Subsequently, the samples were washed with PBS for 24 h, followed by routine dehydration and paraffin embedding. Five-µm sections were cut using a Leica microtome (Leica, Germany) and placed on adhesive-precoated (5% poly-L-lysine) glass slides.

Histological analysis

Histological analysis was performed on 5-µm paraffin sections, stained with hematoxylin and eosin, Masson’s trichrome stain, and specific Safranin O stain. The femur growth plate, growth plate thickness, and chondrocyte numbers were measured using Image J software and corrected by the sample area. Histological sections were viewed under a microscope (Olympus Optical, Olympus BX50) using a 40x objective lens and analyzed using ImageJ. For immunofluorescence analysis, the bone sections were incubated with primary antibodies, including anti-perilipin-1 (Abcam, No. ab3526) and anti-RANKL (Abcam, No. ab45039), followed by incubation with fluorescent dye-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG [1:500, Thermo Fisher Scientific, No. A32731] and Alexa Fluor 555-conjugated anti-mouse IgG [1:500, Thermo Fisher Scientific, No. A31570]). We performed a double-labeling experiment using the TUNEL assay (Millipore, No. S7110), TNFα (Abcam, No. ab1793), AdipoR1 (Abcam; No. ab126611), AdipoR2 (Abcam, No. ab77612), RANKL (Abcam, No.ab45039), and OPG (GeneTex; No. GTX127948). In addition, double immunofluorescence staining was performed in the MC3T3-E1 and RAW264.7 cells using TUNEL (Millipore, No. S7110) staining and LC3 II (Sigma-Aldrich, No. L7643) immunofluorescence staining. The cultured MC3T3-E1 and RAW264.7 cells were incubated with the oxidative fluorescent dye, dihydroethidium (2 µM, Invitrogen, No. D11347). Fluorescent images were captured using a laser scanning confocal microscope system (Carl Zeiss LSM 900, Oberkochen, Germany).

Cell culture and differentiationThe murine calvarial osteoblast cell line MC3T3-E1 subclone 4 (No. CRL-2593) was purchased from ATCC (Manassas, VA) and maintained at 37°C in a humidified 5% CO₂ atmosphere, in ascorbate-free αMEM base media (Gibco, No. A10490-01) supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic solution, and 1 mM sodium pyruvate. The medium was changed three times per week. When the cells reached 80% confluence two or three days after plating, they were subsequently cultured for 0–21 days in differentiation media (ascorbic acid-containing α-MEM, Gibco, No. 12571-063) supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic solution, and 1 mM sodium pyruvate. Cultured cells were analyzed 0, 4, 7, 14, and 21 days after reaching 80% confluence (Suppl. Figure 5a). Mouse-derived macrophages (RAW 264.7) were purchased from ATCC (No. TIB-71) and maintained at 37°C in a humidified 5% CO₂ atmosphere, in a culture medium (DMEM, ATCC, No. 30-2002) supplemented with 10% fetal bovine serum, 100,000 U/L penicillin, and 100 mg/L streptomycin. To induce the differentiation of RAW 264.7 cells into osteoclasts, cells were incubated in an induction medium (ascorbic acid-containing α-MEM) supplemented with 10% fetal bovine serum and 30 ng/mL RANKL (R&D Systems, No. 462-TEC-010/CF) for 7 days. The fresh osteoclast differentiation-inducing medium was changed every 2 days. Thereafter, MC3T3-E1 and RAW264.7 cells were exposed to low glucose (5 mmol/L D-glucose), HG (35 mmol/L D-glucose), PA (500 µM, Sigma-Aldrich), and HG + PA, with or without the additional 48-hour application of AdipoRon (10 nM, AdipoGen Life Sciences, No. AG-CR1-0154). The AccuTarget™ genome-wide predesigned siRNAs targeting AdipoR1 and AdipoR2 and the negative control siRNA (Bioneer Inc., No. SN-1001) were used for transient gene silencing. Transfections of three siRNA mixtures (pooled siRNA reagent) for each gene were performed using Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific, No. 56532), according to the manufacturer’s protocol.

Measurement of cell viability

Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Promega, No. G3580). MC3T3-E1 and RAW 264.7 cells were seeded in a 96-well plate at 2 × 10⁴ cells per well and cultured for 14 and 7 days, respectively (Suppl. Figure 5B). The cells were treated with HG (30 µmol/L), PA (500 µmol/L), or AdipoRon (10 µmol/L) for 48 h. After the incubations, MTT solution was added to each well of a 96-well microplate, and the microplates were placed in an incubator at 37°C for 4 h. Finally, the absorbance was measured at 570 nm using a microplate reader.

Alkaline phosphatase and Alizarin Red S staining assay

MC3T3-E1 cells were seeded at a density of 2 × 10⁴ cells per cm² in 24-well plates to evaluate cell mineralization at five time points (0, 4, 7, 14, and 21 days). At each time point, the cells were fixed in 4% paraformaldehyde and washed three times with PBS. Alkaline phosphatase (ALP) activity and staining were measured using TRACP and ALP assay kits (Takara Bio Inc., No. MK300) according to the manufacturer’s instructions (Suppl. Figure 5a). Calcium deposition was determined by Alizarin Red S (ARS) staining of the MC3T3-E1 cells. The samples were stained with ARS solution (50 mM, pH 4.2; Millipore, No. 2003999) for 30 min at room temperature. After removal of the ARS solution, the samples were washed three times with deionized water and observed under a light microscope (Olympus Optical, Olympus BX50). The calcium deposited in the cells was extracted with 10% cetylpyridinium chloride monohydrate solution for 1 h to quantify the ARS-stained cells. The dye solution was transferred to a 96-well plate, and the absorbance was measured at 550 nm.

Tartrate-resistant acid phosphatase staining assay

RAW264.7 cells were cultured in an osteoclast differentiation-inducing medium containing RANKL (30 ng/mL) for 7 days. They were treated with AdipoRon as described above, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were then incubated with the TRAP staining solution for 30 min. Multinucleated giant cells were counted under a microscope (Olympus Optical, Olympus BX50).

Western blot analysis

Mice bones and cells were extracted using Pro-Prep Protein Extraction Solution (Intron Biotechnology, No. 17081) according to the manufacturer’s instructions. For Nrf2 expression, nuclear proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, No. 78833). Western blotting was performed using the following antibodies: AdipoR1/R2 (Santa Cruz Biotechnology, No. sc-518030 and sc-514045), phospho-Thr¹⁷² AMPK (pAMPK), total AMPK (Cell Signaling Technology, No. 2535S and 2532S), PPARα (Abcam, No. ab215270), PGC–1α (Novus Biologicals, No. NB100-60955), phospho-Ser²⁵⁶ Forkhead box protein O1 (pFOXO1), total FOXO1 (Cell Signaling Technology, No. 9461S and 2880S), LC3 II (Sigma, No. L7543), Perilipin-1/-2 (Abcam, No. ab3526 and ab181452), pACC, total ACC (Santa Cruz, No. SC-271965 and SC-30212), SREBP-1c (Santa Cruz, No. sc-365513), cHREBP1 (Novus Biologicals, No. NB400-135), RUNX2 (Cell Signaling, No. 12556), PPARγ (Abcam, No. ab20935), RANKL (Abcam, No. ab45039), OPG (GeneTex, No. GTX127948), Nrf2 (GeneTex, No. GTX103322), SOD1 (Enzo, No. ADI-SOD-100), SOD2 (Abcam, No. ab16956), NQO1 (Santa Cruz Biotechnology, No. sc-32793), HO-1 (Enzo, No. ADI-OSA-110-F), NOX4 (Santa Cruz Biotechnology, No. sc-30141), CD68 (BD, No. MCA1957), arginase I/II (Santa Cruz Biotechnology, No. sc-271430, sc-393496), iNOS (Novus Biologicals, No. NB300-605), and β-actin (Sigma-Aldrich, No. A5441). After incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling Technology, No. 7076 and 7074), the target proteins were visualized using an enhanced chemiluminescence substrate (ECL Plus; GE Healthcare Bio-Science, No. RPN2106, RPN2232, and RPN2235).

Statistical analysis

The data are expressed as means ± standard deviation. Groups were compared using ANOVA with Bonferroni correction. All statistical analyses were performed with SPSS version 11.5 (SPSS, Chicago, IL, USA). A P-value < 0.05 was considered statistically significant.

Study approval

All animal experiments were performed in accordance with the Laboratory Animals Welfare Act, Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee (IACUC) at the College of Medicine, Catholic University of Korea (CUMC-2017–0251–01).

DATA AVAILABILITY

All data and materials used in this study are displayed or are displayed upon request.

ACKNOWLEDGEMENTS

This study was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (JHL, 2018R1D1A1B07048315, RS-2023-00249560; YK, 2019R1I1A1A01059638, 2022R1I1A1A01065499; CWP, 2019R1H1A2079787, 2020R1F1A1071640, RS-2023-00251436). This study was also supported by the Research Fund of Seoul St. Mary’s Hospital, The Catholic University of Korea (YK, ZC22EISI0876).

CONFLICT OF INTERESTS

The authors have declared that no conflict of interest exists

AUTHOR CONTRIBUTIONS

JHL and CWP designed and conceptualized the experiments. JHL, YK, MYK, ENK, JYP, TWK, HWK, and CWP conducted the experiments and analyzed the data. JHL and YK wrote the manuscript. All authors critically analyzed the manuscript and approved its final version for publication.

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Targeting Bone Marrow Lipid Metabolism with Adiponectin Receptor Agonist for Diabetic and Postmenopausal Osteoporosis

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INTRODUCTION

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DISCUSSION

MATERIALS AND METHODS

Experiment 1

Experiment 2

Biochemical measurements

Microcomputed tomography

Decalcification

Histological analysis

Measurement of cell viability

Alkaline phosphatase and Alizarin Red S staining assay

Tartrate-resistant acid phosphatase staining assay

Western blot analysis

Statistical analysis

Study approval

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