Caspase-2 deficiency protects mice from diabetes-induced marrow adiposity

Type I (T1) diabetes is an autoimmune and metabolic disease associated with bone loss. Bone formation and density are decreased in T1-diabetic mice. Correspondingly, the number of TUNEL positive, dying osteoblasts increases in bones of T1-diabetic mice. Moreover, two known mediators of osteoblast death, TNFα and ROS, are increased in T1-diabetic bone. TNFα and oxidative stress are known to activate caspase-2, a factor involved in the extrinsic apoptotic pathway. Therefore, we investigated the requirement of caspase-2 for diabetes-induced osteoblast death and bone loss. Diabetes was induced in 16-week old C57BL/6 caspase-2 deficient mice and their wild type littermates and markers of osteoblast death, bone formation and resorption, and marrow adiposity were examined. Despite its involvement in extrinsic cell death, deficiency of caspase-2 did not prevent or reduce diabetes-induced osteoblast death as evidenced by a twofold increase in TUNEL positive osteoblasts in both mouse genotypes. Similarly, deficiency of caspase-2 did not prevent T1-diabetes induced bone loss in trabecular bone (BV/TV decreased by 30 and 50%, respectively) and cortical bone (decreased cortical thickness and area with increased marrow area). Interestingly, at this age, differences in bone parameters were not seen between genotypes. However, caspase-2 deficiency attenuated diabetes-induced bone marrow adiposity and adipocyte gene expression. Taken together, our data suggest that caspase-2 deficiency may play a role in promoting marrow adiposity under stress or disease conditions, but it is not required for T1-diabetes induced bone loss.     

Leptin treatment prevents type I diabetic marrow adiposity but not bone loss in mice

Leptin is a hormone secreted by adipocytes that is implicated in the regulation of bone density. Serum leptin levels are decreased in rodent models of type 1 (T1-) diabetes and in diabetic patients. Whether leptin mediates diabetic bone changes is unclear. Therefore, we treated control and T1-diabetic mice with chronic (28 days) subcutaneous infusion of leptin or saline to elucidate the therapeutic potential of leptin for diabetic osteoporosis. Leptin prevented the increase of marrow adipocytes and the increased aP2 expression that we observed in vehicle-treated diabetic mice. However, leptin did not prevent T1-diabetic decreases in trabecular bone volume fraction or bone mineral density in tibia or vertebrae. Consistent with this finding, markers of bone formation (osteocalcin RNA and serum levels) in diabetic mice were not restored to normal levels with leptin treatment. Interestingly, markers of bone resorption (TRAP5 RNA and serum levels) were decreased in diabetic mice by leptin treatment. In summary, we have demonstrated a link between low leptin levels in T1-diabetes and marrow adiposity. However, leptin treatment alone was not successful in preventing bone loss.     

Chronic hyperglycemia modulates osteoblast gene expression through osmotic and non-osmotic pathways

Insulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with osteoporosis and increased fracture rates. The mechanisms underlying IDDM associated bone loss are not known. Previously we demonstrated that osteoblasts exhibit a response to acute (1 and 24 h) hyperglycemia and hyperosmolality. Here we examined the influence of chronic hyperglycemia (30 mM) and its associated hyperosmolality on osteoblast phenotype. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions (mannitol treatment). Acute hyperglycemia for a 48-h period was also capable of inducing alkaline phosphatase and suppressing osteocalcin, MMP-13, VEGF, and GAPDH expression in differentiated osteoblasts. This suggests that acute responses in differentiated cells are maintained chronically. In addition, hyperglycemic and hyperosmotic conditions increased PPARgamma2 expression, although this increase reached significance only in 21 days chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARgamma2 expression is increased in type I diabetic mouse model bones, these findings suggest that diabetes-associated hyperglycemia may modulate osteoblast gene expression, function and bone formation and thereby contribute to type I diabetic bone loss.     

Is insulin an anabolic agent in bone? Dissecting the diabetic bone for clues

Diabetic osteoporosis is increasingly recognized as a significant comorbidity of type 1 diabetes mellitus. In contrast, type 2 diabetes mellitus is more commonly associated with modest increases in bone mineral density for age. Despite this dichotomy, clinical, in vivo, and in vitro data uniformly support the concept that new bone formation as well as bone microarchitectural integrity are altered in the diabetic state, leading to an increased risk for fragility fracture and inadequate bone regeneration following injury. In this review, we examine the contribution that insulin, as a potential anabolic agent in bone, may make to the pathophysiology of diabetic bone disease. Specifically, we have assimilated human and animal data examining the effects of endogenous insulin production, exogenous insulin administration, insulin sensitivity, and insulin signaling on bone. In so doing, we present evidence that insulin, acting as an anabolic agent in bone, can preserve and increase bone density and bone strength, presumably through direct and/or indirect effects on bone formation.     

Loss of interleukin-10 exacerbates early Type-1 diabetes-induced bone loss

Type-1 diabetes (T1D) increases systemic inflammation, bone loss, and risk for bone fractures. Levels of the anti-inflammatory cytokine interleukin-10 (IL-10) are decreased in T1D, however their role in T1D-induced osteoporosis is unknown. To address this, diabetes was induced in male IL-10 knockout (KO) and wild-type (WT) mice. Analyses of femur and vertebral trabecular bone volume fraction identified bone loss in T1D-WT mice at 4 and 12 weeks, which in T1D-IL-10-KO mice was further reduced at 4 weeks but not 12 weeks. IL-10 deficiency also increased the negative effects of T1D on cortical bone. Osteoblast marker osterix was decreased, while osteoclast markers were unchanged, suggesting that IL-10 promotes anabolic processes. MC3T3-E1 osteoblasts cultured under high glucose conditions displayed a decrease in osterix which was prevented by addition of IL-10. Taken together, our results suggest that IL-10 is important for promoting osteoblast maturation and reducing bone loss during early stages of T1D.     

Type I diabetic bone phenotype is location but not gender dependent

Bone is highly dynamic and responsive. Bone location, bone type and gender can influence bone responses (positive, negative or none) and magnitude. Type I diabetes induces bone loss and increased marrow adiposity in the tibia. We tested if this response exhibits gender and location dependency by examining femur, vertebrae and calvaria of male and female, control and diabetic BALB/c mice. Non-diabetic male mice exhibited larger body, muscle, and fat mass, and increased femur BMD compared to female mice, while vertebrae and calvarial bone parameters did not exhibit gender differences. Streptozotocin-induced diabetes caused a reduction in BMD at all sites examined irrespective of gender. Increased marrow adiposity was evident in diabetic femurs and calvaria (endochondrial and intramembranous formed bones, respectively), but not in vertebrae. Leptin-deficient mice also exhibit location dependent bone responses and we found that serum leptin levels were significantly lower in diabetic compared to control mice. However, in contrast to leptin-deficient mice, the vertebrae of T1-diabetic mice exhibit bone loss, not gain. Taken together, our findings indicate that TI-diabetic bone loss in mice is not gender, bone location or bone type dependent, while increased marrow adiposity is location dependent.     

Prominent bone loss mediated by RANKL and IL-17 produced by CD4+ T cells in TallyHo/JngJ mice

Increasing evidence that decreased bone density and increased rates of bone fracture are associated with abnormal metabolic states such as hyperglycemia and insulin resistance indicates that diabetes is a risk factor for osteoporosis. In this study, we observed that TallyHo/JngJ (TH) mice, a polygenic model of type II diabetes, spontaneously developed bone deformities with osteoporotic features. Female and male TH mice significantly gained more body weight than control C57BL/6 mice upon aging. Interestingly, bone density was considerably decreased in male TH mice, which displayed hyperglycemia. The osteoblast-specific bone forming markers osteocalcin and osteoprotegerin were decreased in TH mice, whereas osteoclast-driven bone resorption markers such as IL-6 and RANKL were significantly elevated in the bone marrow and blood of TH mice. In addition, RANKL expression was prominently increased in CD4+ T cells of TH mice upon T cell receptor stimulation, which was in accordance with enhanced IL-17 production. IL-17 production in CD4+ T cells was directly promoted by treatment with leptin while IFN-γ production was not. Moreover, blockade of IFN-γ further increased RANKL expression and IL-17 production in TH-CD4+ T cells. In addition, the osteoporotic phenotype of TH mice was improved by treatment with alendronate. These results strongly indicate that increased leptin in TH mice may act in conjunction with IL-6 to preferentially stimulate IL-17 production in CD4+ T cells and induce RANKL-mediated osteoclastogenesis. Accordingly, we propose that TH mice could constitute a beneficial model for osteoporosis.     

Genetic regulation of metabolic pathways in beta-cells disrupted by hyperglycemia.

In models of type 2 diabetes the expression of beta-cell genes is altered, but these changes have not fully explained the impairment in beta-cell function. We hypothesized that changes in beta-cell phenotype and global alterations in both carbohydrate and lipid pathways are likely to contribute to secretory abnormalities. Therefore, expression of genes involved in carbohydrate and lipid metabolism were analyzed in islets 4 weeks after 85-95% partial pancreatectomy (Px) when beta-cells have impaired glucose-induced insulin secretion and ATP synthesis. Px rats after 1 week developed mild to severe hyperglycemia that was stable for the next 3 weeks, whereas neither plasma triglyceride, non-esterified fatty acid, or islet triglyceride levels were altered. Expression of peroxisome proliferator-activated receptors (PPARs), with several target genes, were reciprocally regulated; PPARalpha was markedly reduced even at low level hyperglycemia, whereas PPARgamma was progressively increased with increasing hyperglycemia. Uncoupling protein 2 (UCP-2) was increased as were other genes barely expressed in sham islets including lactate dehydrogenase-A (LDH-A), lactate (monocarboxylate) transporters, glucose-6-phosphatase, fructose-1,6-bisphosphatase, 12-lipoxygenase, and cyclooxygenase 2. On the other hand, the expression of beta-cell-associated genes, insulin, and GLUT2 were decreased. Treating Px rats with phlorizin normalized hyperglycemia without effecting plasma fatty acids and reversed the changes in gene expression implicating the importance of hyperglycemia per se in the loss of beta-cell phenotype. In addition, parallel changes were observed in beta-cell-enriched tissue dissected by laser capture microdissection from the central core of islets. In conclusion, chronic hyperglycemia leads to a critical loss of beta-cell differentiation with altered expression of genes involved in multiple metabolic pathways diversionary to normal beta-cell glucose metabolism. This global maladaptation in gene expression at the time of increased secretory demand may contribute to the beta-cell dysfunction found in diabetes.     

Protection against T1DM-Induced Bone Loss by Zinc Supplementation: Biomechanical,Histomorphometric, and Molecular Analyses in STZ-Induced Diabetic Rats

Several studies have established an association between diabetes and alterations in bone metabolism; however, the underlying mechanism is not well established. Although zinc is recognized as a potential preventive agent against diabetes-induced bone loss, there is no evidence demonstrating its effect in chronic diabetic conditions. This study evaluated the effects of zinc supplementation in a chronic (90 days) type 1 diabetes-induced bone-loss model. Male Wistar rats were distributed in three groups: control, type 1 diabetes mellitus (T1DM), and T1DM plus zinc supplementation (T1DMS). Serum biochemical analysis; tibia histomorphometric, biomechanical, and collagen-content analyses; and femur mRNA expression were evaluated. Relative to T1DM, the zinc-supplemented group showed increased histomorphometric parameters such as TbWi and BAr and decreased TbSp, increased biomechanical parameters (maximum load, stiffness, ultimate strain, and Young’s modulus), and increased type I collagen content. Interestingly, similar values for these parameters were observed between the T1DMS and control groups. These results demonstrate the protective effect of zinc on the maintenance of bone strength and flexibility. In addition, downregulation of OPG, COL1A, and MMP-9 genes was observed in T1DMS, and the anabolic effects of zinc were evidenced by increased OC expression and serum ALP activity, both related to osteoblastogenesis, demonstrating a positive effect on bone formation. In contrast, T1DM showed excessive bone loss, observed through reduced histomorphometric and biomechanical parameters, characterizing diabetes-associated bone loss. The bone loss was also observed through upregulation of OPG, COL1A, and MMP-9 genes. In conclusion, zinc showed a positive effect on the maintenance of bone architecture and biomechanical parameters. Indeed, OC upregulation and control of expression of OPG, COL1A, and MMP-9 mRNAs, even in chronic hyperglycemia, support an anabolic and protective effect of zinc under chronic diabetic conditions. Furthermore, these results indicate that zinc supplementation could act as a complementary therapy in chronic T1DM.     

Alterations in the growth hormone-insulin-like growth factor axis in insulin dependent diabetes mellitus.

The growth hormone (GH)-insulin-like growth factor (IGF) axis and insulin are major anabolic effectors in promoting weight gain and linear growth. These two anabolic systems are interlinked at many levels, thus abnormalities in one of these systems effect the other causing disordered metabolic homeostasis. Insufficient portal insulinization in insulin dependent diabetes mellitus (IDDM) results in hepatic GH resistance and increased production of IGF-binding proteins-1 (IGFBP-1) and IGFBP-2. GH resistance is reflected by decreased hepatic IGF-I production. In addition, changes in other GH-dependent proteins are also observed in IDDM. Increased proteolysis of IGFBP-3 results in reduction of intact IGFBP-3. Serum ALS levels are also slightly diminished in untreated diabetic patients. Hepatic resistance to GH is, at least in part, caused by diminished GH receptors as reflected by diminished circulating GHBP levels. In addition, there is also evidence from experimental and human studies suggesting post-receptor defect(s) in GH action. As a result of these changes, circulating total and free IGF-I levels are decreased during insulinopenia. Lack of negative feed-back effect of IGF-I on GH secretion causes GH hypersecretion which increases hyperglycemia by decreasing sensitivity to insulin. GH hypersecretion in poorly controlled diabetic patients may play a role in the pathogenesis of diabetic vascular complications. Most of these abnormalities in the GH-IGF axis in diabetes are reversed by effective insulinization of the patient. Addition of IGF-I treatment to insulin in adolescents with IDDM allows correction of GH hypersecretion, improves insulin sensitivity and glycemic control, and decreases insulin requirements. The effect of IGF-I treatment on diabetic complications has yet to be seen.     

Preactivation of β-catenin in osteoblasts improves the osteoanabolic effect of PTH in type 1 diabetic mice

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1–3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.     

Disruption of the insulin-like growth factor type 1 receptor in osteoblasts enhances insulin signaling and action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.     

Amelioration of type I diabetes-induced osteoporosis by parathyroid hormone is associated with improved osteoblast survival

Type 1 diabetic osteoporosis results from impaired osteoblast activity and death. Therefore, anti-resorptive treatments may not effectively treat bone loss in this patient population. Intermittent parathyroid hormone (PTH) treatment stimulates bone remodeling and increases bone density in healthy subjects. However, PTH effects may be limited in patients with diseases that interfere with its signaling. Here, we examined the ability of 8 and 40 μg/kg intermittent PTH to counteract diabetic bone loss. PTH treatment reduced fat pad mass and blood glucose levels in non-diabetic PTH-treated mice, consistent with PTH-affecting glucose homeostasis. However, PTH treatment did not significantly affect general body parameters, including the blood glucose levels, of type 1 diabetic mice. We found that the high dose of PTH significantly increased tibial trabecular bone density parameters in control and diabetic mice, and the lower dose elevated trabecular bone parameters in diabetic mice. The increased bone density was due to increased mineral apposition and osteoblast surface, all of which are defective in type 1 diabetes. PTH treatment suppressed osteoblast apoptosis in diabetic bone, which could further contribute to the bone-enhancing effects. In addition, PTH treatment (40 μg/kg) reversed preexisting bone loss from diabetes. We conclude that intermittent PTH may increase type 1 diabetic trabecular bone volume through its anabolic effects on osteoblasts.     

Synthesis of truncated analogues of preptin-(1–16), and investigation of their ability to stimulate osteoblast proliferation

Preptin, a 34-amino acid residue peptide hormone is co-secreted with insulin from the β-pancreatic cells and is active in fuel metabolism. We have previously established that a shorter fragment of preptin, namely preptin-(1–16), stimulates bone growth by proliferation and increasing the survival rate of osteoblasts. This was demonstrated in both in vitro and in vivo models. These findings suggest that preptin-(1–16) could play an important role in the anabolic therapy of osteoporosis. However, due to the large size of the peptide it is not an ideal therapeutic agent. The aim of this study was to identify the shortest preptin analogue that retains or even increases the bone anabolic activity as compared to the parent preptin-(1–16) peptide. Truncations were made in a methodical manner from both the N-terminus and the C-terminus of the peptide, and the effect of these deletions on the resulting biological activity was assessed. In order to improve the enzymatic stability of the shortest yet active analogue identified, ruthenium-catalysed ring closing metathesis was used to generate a macrocyclic peptide using allylglycine residues as handles for ring formation. We have successfully identified a short 8-amino acid preptin (1–8) fragment that retains an anabolic effect on the proliferation of primary rat osteoblasts and enhances bone nodule formation. Preptin (1–8) is a useful lead compound for the development of orally active therapeutics for the treatment of osteoporosis.     

2型糖尿病与骨质疏松多因素影响的研究进展

摘要：随着人们生活水平的不断提高以及国人寿命的不断延长,2型糖尿病（T2DM）＆骨质疏松（OP）的发病率在全世界范围呈现增高趋势。T2DM并发OP受着性别、年龄、病程、高血糖、糖基化终末产物过多、胰岛素、慢性并发症、肥胖、瘦素、饮食、运动及降糖药物的多种因素影响。骨质疏松症作为糖尿病慢性并发症之一,已严重影响患者的生活质量,T2DM患者在控制血糖同时,应预防其相关因素,定期进行骨密度测定是极为重要的,临床医生应给予高度重视,进行早期预防和治疗。     

Methanolic extract of Cuminum cyminum inhibits ovariectomy-induced bone loss in rats

Several animal and clinical studies have shown that phytoestrogens, plant-derived estrogenic compounds, can be useful in treating postmenopausal osteoporosis. Phytoestrogens and phytoestrogen-containing plants are currently under active investigation for their role in estrogen-related disorders. The present study deals with anti-osteoporotic evaluation of phytoestrogen-rich plant Cuminum cyminum, commonly known as cumin. Adult Sprague-Dawley rats were bilaterally ovariectomized (OVX) and randomly assigned to 3 groups (10 rats/group). Additional 10 animals were sham operated. OVX and sham control groups were orally administered with vehicle while the other two OVX groups were administered 0.15 mg/kg estradiol and 1 g/kg of methanolic extract of Cuminum cyminum fruits (MCC) in two divided doses for 10 weeks. At the end of the study blood, bones and uteri of the animals were collected. Serum was evaluated for calcium, phosphorus, alkaline phosphatase and tartarate resistant acid phosphatase. Bone density, ash density, mineral content and mechanical strength of bones were evaluated. Scanning electron microscopic (SEM) analysis of bones (tibia) was performed. Results were analyzed using ANOVA and Tukeys multiple comparison test. MCC (1 g/kg, p.o.) significantly reduced urinary calcium excretion and significantly increased calcium content and mechanical strength of bones in comparison to OVX control. It showed greater bone and ash densities and improved microarchitecture of bones in SEM analysis. Unlike estradiol it did not affect body weight gain and weight of atrophic uterus in OVX animals. MCC prevented ovariectomy-induced bone loss in rats with no anabolic effect on atrophic uterus. The osteoprotective effect was comparable with estradiol.     

Hyperglycemia-induced oxidative stress in diabetic complications

Reactive oxygen species are increased by hyperglycemia. Hyperglycemia, which occurs during diabetes (both type 1 and type 2) and, to a lesser extent, during insulin resistance, causes oxidative stress. Free fatty acids, which may be elevated during inadequate glycemic control, may also be contributory. In this review, we will discuss the role of oxidative stress in diabetic complications. Oxidative stress may be important in diabetes, not just because of its role in the development of complications, but because persistent hyperglycemia, secondary to insulin resistance, may induce oxidative stress and contribute to beta cell destruction in type 2 diabetes. The focus of this review will be on the role of oxidative stress in the etiology of diabetic complications.     

Effect of chronic lithium on mechanical sensitivity and trabecular bone loss induced by type-1 diabetes mellitus in mice

BioMetals - Type-1 diabetes mellitus (T1DM) is a chronic condition characterized by long-term hyperglycemia that results in several complications such as painful peripheral neuropathy, bone...