Laura R. McCabe

Understanding the pathology and mechanisms of type I diabetic bone loss

Type I (T1) diabetes, also called insulin dependent diabetes mellitus (IDDM), is characterized by little or no insulin production and hyperglycemia. One of the less well known complications of T1‐diabetes is bone loss which occurs in humans and animal models. This complication is receiving increased attention because T1‐diabetics are living longer due to better therapeutics, and are faced with their existing health concerns being compounded by complications associated with aging, such as osteoporosis. Both male and female, endochondrial and intra‐membranous, and axial and appendicular bones are susceptible to T1‐diabetic bone loss. Exact mechanisms accounting for T1‐diabetic bone loss are not known. Existing data indicate that the bone defect in T1‐diabetes is anabolic rather than catabolic, suggesting that anabolic therapeutics may be more effective in preventing bone loss. Potential contributors to T1‐diabetic suppression of bone formation are discussed in this review and include: increased marrow adiposity, hyperlipidemia, reduced insulin signaling, hyperglycemia, inflammation, altered adipokine and endocrine factors, increased cell death, and altered metabolism. Differences between T1‐diabetic‐ and age‐associated bone loss underlie the importance of condition specific, individualized treatments for osteoporosis. Optimizing therapies that prevent bone loss or restore bone density will allow T1‐diabetic patients to live longer with strong healthy bones. J. Cell. Biochem. 102: 1343–1357, 2007.

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 PPARγ2 expression, although this increase reached significance only in 21 days chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARγ2 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. J. Cell. Biochem. 99: 411–424, 2006.

Probiotic L. reuteri Treatment Prevents Bone Loss in a Menopausal Ovariectomized Mouse Model

Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post‐menopausal bone loss. Recent studies suggest an important role for gut‐bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri‐treated mice. Consistent with this, L. reuteri suppressed Ovx‐induced increases in bone marrow CD4+ T‐lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identified that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost‐effective approach to reduce post‐menopausal bone loss. J. Cell. Physiol. 229: 1822–1830, 2014.

Inhibition of PPARγ prevents type I diabetic bone marrow adiposity but not bone loss

Diabetes type I is associated with bone loss and increased bone adiposity. Osteoblasts and adipocytes are both derived from mesenchymal stem cells located in the bone marrow, therefore we hypothesized that if we could block adipocyte differentiation we might prevent bone loss in diabetic mice. Control and insulin‐deficient diabetic BALB/c mice were chronically treated with a peroxisomal proliferator‐activated receptor γ (PPARγ) antagonist, bisphenol‐A‐diglycidyl ether (BADGE), to block adipocyte differentiation. Effects on bone density, adiposity, and gene expression were measured. BADGE treatment did not prevent diabetes‐associated hyperglycemia or weight loss, but did prevent diabetes‐induced hyperlipidemia and effectively blocked diabetes type I‐induced bone adiposity. Despite this, BADGE treatment did not prevent diabetes type I suppression of osteoblast markers (runx2 and osteocalcin) and bone loss (as determined by micro‐computed tomography). BADGE did not suppress osteoblast gene expression or bone mineral density in control mice, however, chronic (but not acute) BADGE treatment did suppress osteocalcin expression in osteoblasts in vitro. Taken together, our findings suggest that BADGE treatment is an effective approach to reduce serum triglyceride and free fatty acid levels as well as bone adiposity associated with type I diabetes. The inability of BADGE treatment to prevent bone loss in diabetic mice suggests that marrow adiposity is not linked to bone density status in type I diabetes, but we cannot exclude the possibility of additional BADGE effects on osteoblasts or other bone cells, which could contribute to preventing the rescue of the bone phenotype. J. Cell. Physiol. 209: 967–976, 2006.

Bone and glucose metabolism: a two-way street.

Evidence from rodent models indicates that undercarboxylated osteocalcin (ucOC), a product of osteoblasts, is a hormone affecting insulin production by the pancreas and insulin sensitivity in peripheral tissues, at least in part through enhanced secretion of adiponectin from adipocytes. Clinical research to test whether this relationship is found in humans is just beginning to emerge. Cross-sectional studies confirm associations between total osteocalcin (OC), ucOC and glucose metabolism but cannot distinguish causality. To date, longitudinal studies have not provided a consistent picture of the effects of ucOC or OC on fasting glucose and insulin sensitivity. Further exploration into the physiological and mechanistic effects of ucOC and OC, in rodent models and clinical studies, is necessary to determine to what extent the skeleton regulates energy metabolism in humans.

Simulated microgravity suppresses osteoblast phenotype, Runx2 levels and AP-1 transactivation.

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood but involve the suppression of osteoblast function. To elucidate the influence of mechanical unloading on osteoblasts, a rotating wall vessel (RWV) was employed as a ground based model of simulated microgravity. Mouse MC3T3‐E1 osteoblasts were grown on microcarrier beads for 14 days and then placed in the RWV for 24 h. Consistent with decreased bone formation during actual spaceflight conditions, alkaline phosphatase and osteocalcin expression were decreased by 80 and 50%, respectively. In addition, runx2 expression and AP‐1 transactivation, key regulators of osteoblast differentiation and bone formation, were reduced by more than 60%. This finding suggests that simulated microgravity could promote dedifferentiation and/or transdifferentiation to alternative cell types; however, markers of adipocyte, chondrocyte, and myocyte lineages were not induced by RWV exposure. Taken together, our results indicate that simulated microgravity may suppress osteoblast differentiation through decreased runx2 and AP‐1 activities.

Extracellular glucose influences osteoblast differentiation and c–jun expression

Insulin dependent diabetes mellitus, marked by high blood glucose levels and no insulin secretion, is associated with decreased bone mass and increased fracture rates. Analysis of bone histology suggests that osteoblast phenotype and function are influenced by diabetes. To determine if elevated extracellular glucose levels could directly influence osteoblast phenotype we treated mouse osteoblasts, MC3T3‐E1 cells, with 22 mM glucose and analyzed osteoblast gene expression. Collagen I mRNA levels significantly increased while osteocalcin mRNA levels decreased 24 h after the addition of glucose. Expression of other genes, actin, osteopontin, and histone H4, was unaffected. Effects on collagen I expression were seen as early as 1 h after treatment. c‐Jun, an AP‐1 transcription factor involved in the regulation of osteoblast gene expression and growth, was also modulated by glucose. Specifically, an increase in c‐jun expression was found at 1 h and maintained for 24 h following glucose treatment. Treatment of osteoblasts with an equal concentration of mannitol completely mimicked glucose treatment effects on collagen I and c‐jun expression, demonstrating that osmotic stress rather than glucose metabolism is responsible for the effects on osteoblast gene expression and phenotype. Additional studies using staurosporine and Ro‐31‐8220 demonstrate that protein kinase C is required for the glucose up regulation of collagen I and c‐jun. Taken together, our results demonstrate that osteoblasts respond to increasing extracellular glucose concentration through an osmotic response pathway that is dependent upon protein kinase C activity and results in upregulation of c‐jun and modulation of collagen I and osteocalcin expression. J. Cell. Biochem. 79:301–310, 2000.

Streptozotocin, Type I Diabetes Severity and Bone

As many as 50% of adults with type I (T1) diabetes exhibit bone loss and are at increased risk for fractures. Therapeutic development to prevent bone loss and/or restore lost bone in T1 diabetic patients requires knowledge of the molecular mechanisms accounting for the bone pathology. Because cell culture models alone cannot fully address the systemic/metabolic complexity of T1 diabetes, animal models are critical. A variety of models exist including spontaneous and pharmacologically induced T1 diabetic rodents. In this paper, we discuss the streptozotocin (STZ)-induced T1 diabetic mouse model and examine dose-dependent effects on disease severity and bone. Five daily injections of either 40 or 60 mg/kg STZ induce bone pathologies similar to spontaneously diabetic mouse and rat models and to human T1 diabetic bone pathology. Specifically, bone volume, mineral apposition rate, and osteocalcin serum and tibia messenger RNA levels are decreased. In contrast, bone marrow adiposity and aP2 expression are increased with either dose. However, high-dose STZ caused a more rapid elevation of blood glucose levels and a greater magnitude of change in body mass, fat pad mass, and bone gene expression (osteocalcin, aP2). An increase in cathepsin K and in the ratio of RANKL/OPG was noted in high-dose STZ mice, suggesting the possibility that severe diabetes could increase osteoclast activity, something not seen with lower doses. This may contribute to some of the disparity between existing studies regarding the role of osteoclasts in diabetic bone pathology. Examination of kidney and liver toxicity indicate that the high STZ dose causes some liver inflammation. In summary, the multiple low-dose STZ mouse model exhibits a similar bone phenotype to spontaneous models, has low toxicity, and serves as a useful tool for examining mechanisms of T1 diabetic bone loss.

Human bone marrow adiposity is linked with serum lipid levels not T1-diabetes.

Increased marrow adiposity is often associated with bone loss. Little is known about the regulation of marrow adiposity in humans. Marrow adiposity is increased in several mouse models including type I (T1)-diabetic mice, which also display bone loss. However, the impact of metabolic disease on marrow adiposity in humans has yet to be examined. This study measured bone marrow adiposity levels with iterative decomposition of water and fat with echo asymmetry and least-squares estimation magnetic resonance imaging and determined their relationship with T1-diabetes, bone mineral density (BMD), and serum lipid levels. Participants were adult T1-diabetic patients (glycosylated hemoglobin averaging 7.70%±0.4%) and age- and body-mass-index-matched nondiabetic subjects. Consistent with previous reports, serum osteocalcin levels were lower in subjects with T1-diabetes compared to controls (reaching statistical significance in females) and negatively correlated with disease duration (r=-0.50, P<.01). Furthermore, femur neck BMD inversely correlated with diabetes severity (r=-0.417, P<.05). While marrow adiposity was not altered by T1-diabetes, there was a striking positive correlation between vertebral, femur, and tibia marrow adiposity and serum lipid levels (low-density lipoprotein, total cholesterol, cholesterol:high-density lipoprotein ratio, and triglyceride; r≥0.383), reaching a significance of P<.001 in some comparisons. Marrow adiposity also displayed strong intrasubject correlations at multiple bone sites (r≥0.411, P<.05), increased with age (r=0.410, P<.05 at vertebral sites), and was reciprocally related to bone density (r≥-0.378, P<.05). Taken together, our data suggest that marrow adiposity may be an indicator of elevated serum lipid levels and decreased bone density.

The bone marrow microenvironment contributes to type I diabetes induced osteoblast death

Type I diabetes increases an individuals risk for bone loss and fracture, predominantly through suppression of osteoblast activity (bone formation). During diabetes onset, levels of blood glucose and pro‐inflammatory cytokines (including tumor necrosis factor α (TNFα)) increased. At the same time, levels of osteoblast markers are rapidly decreased and stay decreased chronically (i.e., 40 days later) at which point bone loss is clearly evident. We hypothesized that early bone marrow inflammation can promote osteoblast death and hence reduced osteoblast markers. Indeed, examination of type I diabetic mouse bones demonstrates a greater than twofold increase in osteoblast TUNEL staining and increased expression of pro‐apoptotic factors. Osteoblast death was amplified in both pharmacologic and spontaneous diabetic mouse models. Given the known signaling and inter‐relationships between marrow cells and osteoblasts, we examined the role of diabetic marrow in causing the osteoblast death. Co‐culture studies demonstrate that compared to control marrow cells, diabetic bone marrow cells increase osteoblast (MC3T3 and bone marrow derived) caspase 3 activity and the ratio of Bax/Bcl‐2 expression. Mouse blood glucose levels positively correlated with bone marrow induced osteoblast death and negatively correlated with osteocalcin expression in bone, suggesting a relationship between type I diabetes, bone marrow and osteoblast death. TNF expression was elevated in diabetic marrow (but not co‐cultured osteoblasts); therefore, we treated co‐cultures with TNFα neutralizing antibodies. The antibody protected osteoblasts from bone marrow induced death. Taken together, our findings implicate the bone marrow microenvironment and TNFα in mediating osteoblast death and contributing to type I diabetic bone loss. J. Cell. Physiol. 226: 477–483, 2011.