Hyperbaric oxygen therapy exerts anti-osteoporotic effects in obese and lean D-galactose-induced aged rats.

Obesity accelerates the aging processes, resulting in an aggravation of aging-induced osteoporosis. We investigated the anti-osteoporotic effect of hyperbaric oxygen therapy (HBOT) in obese- and lean-aged rats through measurement of cellular senescence, hypoxia, inflammation, antioxidants, and bone microarchitecture. Obese and lean male Wistar rats were injected with 150 mg/kg/day of D-galactose for 8 weeks to induce aging. Then, all rats were randomly given either sham or HBOT for 14 days. Metabolic parameters were determined. Expression by bone mRNA for cellular senescence, hypoxia, inflammation, antioxidative capacity, and bone remodeling were examined. Micro-computed tomography and atomic absorption spectroscopy were performed to evaluate bone microarchitecture and bone mineral profiles, respectively. We found that HBOT restored the alterations in the mRNA expression level of p16, p21, HIF-1α, TNF-α, IL-6, RANKL, RANK, NFATc1, DC-STAMP, Osx, ALP, and Col1a1 in the bone in obese-and lean- aging rats. In obese-aging rats, HBOT increased the level of expression of Sirt1 and CuZnSOD mRNA and diminished the expression level of HIF-2α and ctsk mRNA to the same levels as the control group. However, HBOT failed to alter catalase and OCN mRNA expression in obese-aged rats. HBOT partially improved the bone microarchitecture in obese-aged rats, but completely restored it in lean-aged rats. Interestingly, HBOT protected against obesity-induced demineralization in obese-aged rats. In summary, HBOT exerts an anti-osteoporotic effect in lean-aged rats and prevents some, but not all the negative effects of obese-aged conditions on bone health. Therefore, HBOT is considered as a potential therapy for aging-induced osteoporosis, regardless of obese status.

Parathyroid hormone increases CFTR expression and function in Caco-2 intestinal epithelial cells.

Parathyroid hormone (PTH) enhances cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion secretion by the human intestinal epithelial cell line Caco-2. With the patch-clamp and Ussing chamber techniques, we investigated how PTH stimulates CFTR activity in Caco-2 cells. Cell-attached recordings revealed that PTH stimulated the opening of CFTR-like channels, while impedance analysis demonstrated that PTH increased apical membrane capacitance, a measure of membrane surface area. Using ion substitution experiments, the PTH-stimulated increase in short-circuit current (Isc), a measure of transepithelial ion transport, was demonstrated to be Cl-- and HCO3--dependent. However, the PTH-stimulated increase in Isc was unaffected by the carbonic anhydrase inhibitor acetazolamide, but partially blocked by the intermediate-conductance Ca2+-activated K+ channel (IKCa) inhibitor clotrimazole. TRAM-34, a related IKCa inhibitor, failed to directly inhibit CFTR Cl- channels in cell-free membrane patches, excluding its action on CFTR. In conclusion, PTH enhances CFTR-mediated anion secretion by Caco-2 monolayers by increasing the expression and function of CFTR in the apical membrane and IKCa activity in the basolateral membrane.

Anomalous bone changes in ovariectomized type 2 diabetic rats: inappropriately low bone turnover with bone loss in an estrogen-deficient condition.

Estrogen deprivation accelerates bone resorption, leading to imbalance of bone remodeling and osteoporosis in postmenopausal women. In the elderly, type 2 diabetes mellitus (T2DM) frequently coexists as an independent factor of bone loss. However, little is known about the skeletal changes in a combined condition of estrogen deficiency and T2DM. Herein, we performed ovariectomy (OVX) in nonobese Goto-Kakizaki (GK) T2DM rats to examine changes associated with calcium and phosphate metabolism and bone microstructures and strength. As expected, wild-type (WT) rats subjected to ovariectomy (OVX-WT) had low trabecular bone volume and serum calcium with increased dynamic histomorphometric and serum bone markers, consistent with the high turnover state. T2DM in GK rats also led to low trabecular volume and serum calcium. However, the dynamic histomorphometric markers of bone remodeling were unaffected in these GK rats, indicating the distinct mechanism of T2DM-induced bone loss. Interestingly, OVX-GK rats were found to have anomalous and unique changes in bone turnover-related parameters, i.e., decreased osteoblast and osteoclast surfaces with lower COOH-terminal telopeptide of type I collagen levels compared with OVX-WT rats. Furthermore, the levels of calciotropic hormones, i.e., parathyroid hormone and 1,25(OH)2D3, were significantly decreased in OVX-GK rats. Although the OVX-induced bone loss did not further worsen in GK rats, a three-point bending test indicated that OVX-GK bones exhibited a decrease in bone elasticity. In conclusion, T2DM and estrogen deficiency both led to microstructural bone loss, the appearance of which did not differ from each factor alone. Nevertheless, the combination worsened the integrity and suppressed the turnover, which might eventually result in adynamic bone disease.

Premature chondrocyte apoptosis and compensatory upregulation of chondroregulatory protein expression in the growth plate of Goto-Kakizaki diabetic rats.

Type 2 diabetes mellitus (T2DM) is much more detrimental to bone than previously thought. Specifically, it is associated with aberrant bone remodeling, defective bone microstructure, poor bone quality, and growth retardation. The T2DM-associated impairment of bone elongation may result from a decrease in growth plate function, but the detailed mechanism has been unknown. The present study, therefore, aimed to test hypothesis that T2DM led to premature apoptosis of growth plate chondrocytes in Goto-Kakizaki (GK) type 2 diabetic rats, and thus triggered the compensatory responses to overcome this premature apoptosis, such as overexpression of Runt-related transcription factor (Runx)-2 and vascular endothelial growth factor (VEGF), the essential mediators for bone elongation. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of epiphyseal sections successfully revealed increases in chondrocyte apoptosis in the hypertrophic zone (HZ) and chondro-osseous junction of GK rats. Quantitative immunohistochemical analysis further confirmed the overexpression of parathyroid hormone-related protein (PTHrP), Runx2 and VEGF, but not Indian hedgehog (Ihh) in the HZ. Analysis of blood chemistry indicated suppression of bone remodeling with a marked decrease in parathyroid hormone level. In conclusion, GK rats manifested a premature increase in chondrocyte apoptosis in the HZ of growth plate, and a compensatory overexpression of chondroregulatory proteins, such as PTHrP, Runx2, and VEGF. Our results, therefore, help explain how T2DM leads to impaired bone elongation and growth retardation.

Fe3+ opposes the 1,25(OH)2D3-induced calcium transport across intestinal epithelium-like Caco-2 monolayer in the presence or absence of ascorbic acid.

Although iron is an essential element for hemoglobin and cytochrome synthesis, excessive intestinal iron absorption-as seen in dietary iron supplementation and hereditary disease called thalassemia-could interfere with transepithelial transport of calcium across the intestinal mucosa. The underlying cellular mechanism of iron-induced decrease in intestinal calcium absorption remains elusive, but it has been hypothesized that excess iron probably negates the actions of 1,25-dihydroxyvitamin D [1,25(OH)2D3]. Herein, we exposed the 1,25(OH)2D3-treated epithelium-like Caco-2 monolayer to FeCl3 to demonstrate the inhibitory effect of ferric ion on 1,25(OH)2D3-induced transepithelial calcium transport. We found that a 24-h exposure to FeCl3 on the apical side significantly decreased calcium transport, while increasing the transepithelial resistance (TER) in 1,25(OH)2D3-treated monolayer. The inhibitory action of FeCl3 was considered rapid since 60-min exposure was sufficient to block the 1,25(OH)2D3-induced decrease in TER and increase in calcium flux. Interestingly, FeCl3 did not affect the baseline calcium transport in the absence of 1,25(OH)2D3 treatment. Furthermore, although ascorbic acid is often administered to maximize calcium solubility and to enhance intestinal calcium absorption, it apparently had no effect on calcium transport across the FeCl3- and 1,25(OH)2D3-treated Caco-2 monolayer. In conclusion, apical exposure to ferric ion appeared to negate the 1,25(OH)2D3-stimulated calcium transport across the intestinal epithelium. The present finding has, therefore, provided important information for development of calcium and iron supplement products and treatment protocol for specific groups of individuals, such as thalassemia patients and pregnant women.

Vasoactive intestinal peptide and cystic fibrosis transmembrane conductance regulator contribute to the transepithelial calcium transport across intestinal epithelium-like Caco-2 monolayer.

Vasoactive intestinal peptide (VIP) as a neurocrine factor released by enteric neurons has been postulated to participate in the regulation of transcellular active calcium transport across intestinal epithelium, but the preceding evidence is scant and inconclusive. Herein, transepithelial calcium flux and epithelial electrical parameters were determined by Ussing chamber technique with radioactive tracer in the intestinal epithelium-like Caco-2 monolayer grown on Snapwell. After 3-day culture, Caco-2 cells expressed mRNA of calcium transporters, i.e., TRPV6, calbindin-D9k, PMCA1b and NCX1, and exhibited transepithelial resistance of ~200 Ω cm2, a characteristic of leaky epithelium similar to the small intestine. VIP receptor agonist was able to enhance transcellular calcium flux, whereas VIP receptor antagonist totally abolished calcium fluxes induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Since the intestinal cystic fibrosis transmembrane conductance regulator (CFTR) could be activated by VIP and calciotropic hormones, particularly parathyroid hormone, we sought to determine whether CFTR also contributed to the 1,25(OH)2D3-induced calcium transport. A selective CFTR inhibitor (20-200 µM CFTRinh-172) appeared to diminish calcium fluxes as well as transepithelial potential difference and short-circuit current, both of which indicated a decrease in electrogenic ion transport. On the other hand, 50 µM genistein-a molecule that could rapidly activate CFTR-was found to increase calcium transport. Our in silico molecular docking analysis confirmed direct binding of CFTRinh-172 and genistein to CFTR channels. In conclusion, VIP and CFTR apparently contributed to the intestinal calcium transport, especially in the presence of 1,25(OH)2D3, thereby supporting the existence of the neurocrine control of intestinal calcium absorption.

D-galactose-induced aging aggravates obesity-induced bone dyshomeostasis.

We aimed to compare the time-course effect of D-galactose (D-gal)-induced aging, obesity, and their combined effects on bone homeostasis. Male Wistar rats were fed with either a normal diet (ND; n = 24) or a high-fat diet (HFD; n = 24) for 12 weeks. All rats were then injected with either vehicle or 150 mg/kg/day of D-gal for 4 or 8 weeks. Blood was collected to measure metabolic, aging, oxidative stress, and bone turnover parameters. Bone oxidative stress and inflammatory markers, as well as bone histomorphometry were also evaluated. Additionally, RAW 264.7 cells were incubated with either D-gal, insulin, or D-gal plus insulin to identify osteoclast differentiation capacity under the stimulation of receptor activator of nuclear factor κB ligand. At week 4, D-gal-induced aging significantly elevated serum malondialdehyde level and decreased trabecular thickness in ND- and HFD-fed rats, when compared to the control group. At week 8, D-gal-induced aging further elevated advanced glycation end products, increased bone inflammation and resorption, and significantly impaired bone microarchitecture in HFD-fed rats. The osteoclast number in vitro were increased in the D-gal, insulin, and combined groups to a similar extent. These findings suggest that aging aggravates bone dyshomeostasis in the obese condition in a time-dependent manner.

Hyperbaric oxygen therapy improves age induced bone dyshomeostasis in non-obese and obese conditions.

AIMS: To investigate the effects of hyperbaric oxygen therapy (HBOT) on metabolic disturbance, aging and bone remodeling in D-galactose-induced aging rats with and without obesity by determining the metabolic parameters, aging and oxidative stress markers, bone turnover markers, bone microarchitecture, and bone biomechanical strength. MATERIALS AND METHODS: Male Wistar rats were fed either a normal diet (ND; n = 18) or a HFD (n = 12) for 22 weeks. At week 13, vehicle (0.9% NaCl) was injected into ND-fed rats (NDV; n = 6), while 150 mg/kg/day of D-galactose was injected into 12 ND-fed rats (NDD) and 12 HFD-fed rats (HFDD) for 10 weeks. At week 21, rats were treated with either sham (NDVS, NDDS, or HFDDS; n = 6/ group) or HBOT (NDDH, or HFDDH; n = 6/group) for 14 days. Rats were then euthanized. Blood samples, femora, and tibiae were collected. KEY FINDINGS: Both NDD and HFDD groups developed aging as indicated by increased AGE level, increased inflammation and oxidative stress as shown by raised serum TNF-α and MDA levels, impaired bone remodeling as indicated by an increase in levels of CTX-1, TRACP-5b, and impaired bone structure/strength, when compared with those of the NDVS group. HFD aggravated these indicators of bone dyshomeostasis in D-galactose-treated rats. HBOT restored bone remodeling and bone structure/strength in the NDD group, however HBOT ameliorated bone dyshomeostasis in the HFDD group. SIGNIFICANCE: HBOT is a potential intervention to decrease the risk of osteoporosis and bone fracture in aging with or without obesity.

Mild-intensity physical activity prevents cardiac and osseous iron deposition without affecting bone mechanical property or porosity in thalassemic mice.

Thalassemia causes anemia, ineffective erythropoiesis, bone loss and iron accumulation in several tissues, e.g., liver, bone and heart, the last of which leads to lethal cardiomyopathy and arrhythmia. Although exercise reportedly improves bone density in thalassemic mice, exercise performance is compromised and might pose risk of cardiovascular accident in thalassemic patients. Therefore, we sought to explore whether mild-intensity physical activity (MPA) with 30-50% of maximal oxygen consumption was sufficient to benefit the heart and bone. Herein, male hemizygous ß-globin knockout (BKO) mice and wild-type littermates were subjected to voluntary wheel running 1 h/day, 5 days/week for 3 months (MPA group) or kept sedentary (SDN; control). As determined by atomic absorption spectroscopy, BKO-MPA mice had less iron accumulation in heart and bone tissues compared with BKO-SDN mice. Meanwhile, the circulating level of fibroblast growth factor-23-a factor known to reduce serum iron and intestinal calcium absorption-was increased early in young BKO-MPA mice. Nevertheless, MPA did not affect duodenal calcium transport or body calcium retention. Although MPA restored the aberrant bone calcium-phosphorus ratio to normal range, it did not change vertebral calcium content or femoral mechanical properties. Microstructural porosity in tibia of BKO-MPA mice remained unaltered as determined by synchrotron radiation X-ray tomographic microscopy. In conclusion, MPA prevents cardiac and bone iron accumulation, which is beneficial to thalassemic patients with limited physical fitness or deteriorated cardiac performance. However, in contrast to moderate-intensity exercise, MPA does not improve bone mechanical properties or reduce bone porosity.

Excessive salt consumption causes systemic calcium mishandling and worsens microarchitecture and strength of long bones in rats.

Excessive salt intake has been associated with the development of non-communicable diseases, including hypertension with several cardiovascular consequences. Although the detrimental effects of high salt on the skeleton have been reported, longitudinal assessment of calcium balance together with changes in bone microarchitecture and strength under salt loading has not been fully demonstrated. To address these unanswered issues, male Sprague-Dawley rats were fed normal salt diet (NSD; 0.8% NaCl) or high salt diet (HSD; 8% NaCl) for 5 months. Elevation of blood pressure, cardiac hypertrophy and glomerular deterioration were observed in HSD, thus validating the model. The balance studies were performed to monitor calcium input and output upon HSD challenge. The HSD-induced increase in calcium losses in urine and feces together with reduced fractional calcium absorption led to a decrease in calcium retention. With these calcium imbalances, we therefore examined microstructural changes of long bones of the hind limbs. Using the synchrotron radiation x-ray tomographic microscopy, we showed that trabecular structure of tibia and femur of HSD displayed a marked increase in porosity. Consistently, the volumetric micro-computed tomography also demonstrated a significant decrease in trabecular bone mineral density with expansion of endosteal perimeter in the tibia. Interestingly, bone histomorphometric analyses indicated that salt loading caused an increase in osteoclast number together with decreases in osteoblast number and osteoid volume. This uncoupling process of bone remodeling in HSD might underlie an accelerated bone loss and bone structural changes. In conclusion, long-term excessive salt consumption leads to impairment of skeletal mass and integrity possibly through negative calcium balance.

Responses of primary osteoblasts and osteoclasts from hemizygous ß-globin knockout thalassemic mice with elevated plasma glucose to 1,25-dihydroxyvitamin D3.

ß-thalassemia is often associated with hyperglycemia, osteoporosis and increased fracture risk. However, the underlying mechanisms of the thalassemia-associated bone loss remain unclear. It might result from abnormal activities of osteoblasts and osteoclasts, and perhaps prolonged exposure to high extracellular glucose. Herein, we determined the rate of duodenal calcium transport in hemizygous ß-globin knockout thalassemic (BKO) mice. Their bones were collected for primary osteoblast and osteoclast culture. We found that BKO mice had lower calcium absorption than their wild-type (WT) littermates. Osteoblasts from BKO mice showed aberrant expression of osteoblast-specific genes, e.g., Runx2, alkaline phosphatase and osteocalcin, which could be partially restored by 1,25(OH)2D3 treatment. However, the mRNA expression levels of RANK, calcitonin receptor (Calcr), c-Fos, NFATc1, cathepsin K and DMT1 were similar in both BKO and WT groups. Exposure to high extracellular glucose modestly but significantly affected the expression of osteoclast-specific markers in WT osteoclasts with no significant effect on osteoblast-specific genes in WT osteoblasts. Thus, high glucose alone was unable to convert WT bone cells to BKO-like bone cells. In conclusion, the impaired calcium absorption and mutation-related aberrant bone cell function rather than exposure to high blood glucose were likely to be the principal causes of thalassemic bone loss.

Dipeptidyl Peptidase-4 Inhibitor, Vildagliptin, Improves Trabecular Bone Mineral Density and Microstructure in Obese, Insulin-Resistant, Pre-diabetic Rats.

OBJECTIVE: Obese insulin resistance and type 2 diabetes mellitus profoundly impair bone mechanical properties and bone quality. However, because several antidiabetes drugs, especially thiazolidinediones, further aggravate bone loss in individuals with diabetes, diabetic osteopathy should not be treated by using simply any glucose-lowering agents. Recently, incretins have been reported to affect osteoblast function positively. The present study aimed to investigate the effects of vildagliptin, an inhibitor of dipeptidyl peptidase-4, on bone of rats with high-fat-diet-induced prediabetes. METHODS: Male rats were fed a high-fat diet for 12 weeks to induce obese insulin resistance and then treated with vildagliptin for 4 weeks. The effects of the drug on bone were determined by microcomputed tomography and bone histomorphometry. RESULTS: Vildagliptin markedly improved insulin resistance in these obese insulin-resistant rats. It also significantly increased volumetric bone mineral density. Specifically, vildagliptin-treated obese insulin-resistant rats exhibited higher trabecular volumetric bone mineral density than vehicle-treated obese insulin-resistant rats, whereas cortical volumetric bone mineral density, cortical thickness and area were not changed. Bone histomorphometric analysis in a trabecular-rich area (i.e. tibial metaphysis) revealed greater trabecular bone volume and number and less trabecular separation without change in trabecular thickness, osteocyte lacunar area or cortical thickness in the vildagliptin-treated group. CONCLUSIONS: Vildagliptin had a beneficial effect on the bone of obese insulin-resistant rats with prediabetes, particularly at the trabecular site. Such benefit probably results from enhanced bone formation rather than from suppressed bone resorption.

Insulin does not rescue cortical and trabecular bone loss in type 2 diabetic Goto-Kakizaki rats.

In type 2 diabetes mellitus (T2DM), the decreased bone strength is often associated with hyperglycemia and bone cell insulin resistance. Since T2DM is increasingly reported in young adults, it is not known whether the effect of T2DM on bone would be different in young adolescents and aging adults. Here, we found shorter femoral and tibial lengths in 7-month, but not 13-month, Goto-Kakizaki (GK) T2DM rats as compared to wild-type rats. Bone µCT analysis showed long-lasting impairment of both cortical and trabecular bones in GK rats. Although insulin treatment effectively improved hyperglycemia, it was not able to rescue trabecular BMD and cortical thickness in young adult GK rats. In conclusion, insulin treatment and alleviation of hyperglycemia did not increase BMD of osteopenic GK rats. It is likely that early prevention of insulin resistance should prevail over treatment of full-blown T2DM-related osteopathy.

Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-κB pathway.

Cyperenoic acid is a terpenoid isolated from the root of a medicinal plant Croton crassifolius with a wide range of biological activities. In this study, the effects of cyperenoic acid on osteoclast differentiation were investigated both in vitro and in vivo using receptor activator of nuclear factor-κB ligand (RANKL)-induced bone marrow-derived osteoclasts and senescence-accelerated mouse prone 6 (SAMP6). Cyperenoic acid significantly suppressed RANKL-induced osteoclast differentiation at the concentrations with no apparent cytotoxicity. The half maximum inhibitory concentration (IC50) for osteoclast differentiation was 36.69 µM ± 1.02. Cyperenoic acid treatment evidently reduced the expression of two key transcription factors in osteoclast differentiation, NFATc1 and c-Fos. Detailed signaling analysis revealed that cyperenoic acid did not affect MAPK pathways and canonical NF-κB pathway but impaired activation of p100/p52 in the non-canonical NF-κB pathway upon RANKL stimulation. Moreover, the expression of osteoclast-related genes, nfatc1, ctsk, irf8, acp5 and cfos were disrupted by cyperenoic acid treatment. The bone resorption activity by cyperenoic acid-treated osteoclasts were impaired. In a senile osteoporosis mouse model SAMP6, mice fed on diet supplemented with cyperenoic acid showed delay in bone loss, compared to the control. Taken together, plant-derived cyperenoic acid shows great potential as therapeutic agent for osteoporosis.

Estrogen deprivation aggravates cardiac hypertrophy in nonobese Type 2 diabetic Goto-Kakizaki (GK) rats.

Both Type 2 diabetes mellitus (T2DM) and estrogen deprivation have been shown to be associated with the development of cardiovascular disease and adverse cardiac remodeling. However, the role of estrogen deprivation on adverse cardiac remodeling in nonobese T2DM rats has not been clearly elucidated. We hypothesized that estrogen-deprivation aggravates adverse cardiac remodeling in Goto-Kakizaki (GK) rats. Wild-type (WT) and GK rats at the age of 9 months old were divided into two subgroups to have either a sham operation (WTS, GKS) or a bilateral ovariectomy (WTO, GKO) (n = 6/subgroup). Four months after the operation, the rats were killed, and the heart was excised rapidly. Metabolic parameters, cardiomyocytes hypertrophy, cardiac fibrosis, and biochemical parameters were determined. GK rats had hyperglycemia with hypoinsulinemia, and estrogen deprivation did not increase the severity of T2DM. Cardiac hypertrophy, cardiac oxidative stress, and phosphor-antinuclear factor κB were higher in WTO and GKS rats than WTS rats, and they markedly increased in GKO rats compared with GKS rats. Furthermore, cardiac fibrosis, transforming growth factor-ß, Bax, phosphor-p38, and peroxisome proliferator- activated receptor γ coactivator-1α expression were increased in GKS and GKO rats compared with the lean rats. However, mitochondrial dynamics proteins including dynamin-related protein 1 and mitofusin-2 were not altered by T2DM and estrogen deprivation. Although estrogen deprivation did not aggravate T2DM in GK rats, it increased the severity of cardiac hypertrophy by provoking cardiac inflammation and oxidative stress in nonobese GK rats.

High dietary cholesterol masks type 2 diabetes-induced osteopenia and changes in bone microstructure in rats.

Type 2 diabetes mellitus (T2DM) often occurs concurrently with high blood cholesterol or dyslipidemia. Although T2DM has been hypothesized to impair bone microstructure, several investigations showed that, when compared to age-matched healthy individuals, T2DM patients had normal or relatively high bone mineral density (BMD). Since cholesterol and lipids profoundly affect the function of osteoblasts and osteoclasts, it might be cholesterol that obscured the changes in BMD and bone microstructure in T2DM. The present study, therefore, aimed to determine bone elongation, epiphyseal histology, and bone microstructure in non-obese T2DM Goto-Kakizaki rats treated with normal (GK-ND) and high cholesterol diet. We found that volumetric BMD was lower in GK-ND rats than the age-matched wild-type controls. In histomorphometric study of tibial metaphysis, T2DM evidently suppressed osteoblast function as indicated by decreases in osteoblast surface, mineral apposition rate, and bone formation rate in GK-ND rats. Meanwhile, the osteoclast surface and eroded surface were increased in GK-ND rats, thus suggesting an activation of bone resorption. T2DM also impaired bone elongation, presumably by retaining the chondrogenic precursor cells in the epiphyseal resting zone. Interestingly, several bone changes in GK rats (e.g., increased osteoclast surface) disappeared after high cholesterol treatment as compared to wild-type rats fed high cholesterol diet. In conclusion, high cholesterol diet was capable of masking the T2DM-induced osteopenia and changes in several histomorphometric parameters that indicated bone microstructural defect. Cholesterol thus explained, in part, why a decrease in BMD was not observed in T2DM, and hence delayed diagnosis of the T2DM-associated bone disease.