Development and Application of Mass Spectroscopy Assays for Nε-(1-Carboxymethyl)-L-Lysine and Pentosidine in Renal Failure and Diabetes.

BACKGROUND: Advanced glycation end products (AGEs) are formed via the nonenzymatic glycation of sugars with amino acids. Two AGEs, Nε-(1-carboxymethyl)-L-Lysine (CML) and pentosidine, have been observed to be elevated in subjects suffering from a multitude of chronic disease states, and accumulation of these compounds may be related to the pathophysiology of disease progression and aging. METHODS: We describe here the development and validation of a specific and reproducible LC-MS/MS method to quantify CML and pentosidine in human serum with lower limits of quantitation of 75 ng/mL and 5 ng/mL, respectively. The analyte calibration curve exhibited excellent linearity at a range of 0-10 900 ng/mL for CML and 0-800 ng/mL for pentosidine. High-low linearity of 5 serum pairs was assessed, with a mean recovery of 103% (range 94-116%) for CML, and 104% (range 97-116%) for pentosidine. RESULTS: Serum concentrations of CML and pentosidine were quantified in 30 control and 30 subjects with chronic renal insufficiency. A significant increase in both analytes was observed in renal failure compared to control subjects (2.1-fold and 8.4-fold, respectively; P < 0.001 for both). In a separate cohort of 49 control versus 95 subjects with type 2 diabetes mellitus (T2DM), serum CML but not serum pentosidine, was significantly elevated in the T2DM patients, and CML was also correlated with glycemic control, as assessed by hemoglobin A1c (r = 0.34, P < 0.001). CONCLUSIONS: These mass spectroscopy-based assays for serum CML and pentosidine should be useful in accurately evaluating circulating levels of these key AGEs in various disease states.

Accelerated osteocyte senescence and skeletal fragility in mice with type 2 diabetes.

The worldwide prevalence of type 2 diabetes (T2D) is increasing. Despite normal to higher bone density, patients with T2D paradoxically have elevated fracture risk resulting, in part, from poor bone quality. Advanced glycation endproducts (AGEs) and inflammation as a consequence of enhanced receptor for AGE (RAGE) signaling are hypothesized culprits, although the exact mechanisms underlying skeletal dysfunction in T2D are unclear. Lack of inducible models that permit environmental (in obesity) and temporal (after skeletal maturity) control of T2D onset has hampered progress. Here, we show in C57BL/6 mice that a onetime pharmacological intervention (streptozotocin, STZ) initiated in adulthood combined with high-fat diet-induced (HFD-induced) obesity caused hallmark features of human adult-onset T2D, including prolonged hyperglycemia, insulin resistance, and pancreatic ß cell dysfunction, but not complete destruction. In addition, HFD/STZ (i.e., T2D) resulted in several changes in bone quality that closely mirror those observed in humans, including compromised bone microarchitecture, reduced biomechanical strength, impaired bone material properties, altered bone turnover, and elevated levels of the AGE CML in bone and blood. Furthermore, T2D led to the premature accumulation of senescent osteocytes with a unique proinflammatory signature. These findings highlight the RAGE pathway and senescent cells as potential targets to treat diabetic skeletal fragility.

Independent Roles of Estrogen Deficiency and Cellular Senescence in the Pathogenesis of Osteoporosis: Evidence in Young Adult Mice and Older Humans.

Estrogen deficiency is a seminal mechanism in the pathogenesis of osteoporosis. Mounting evidence, however, establishes that cellular senescence, a fundamental mechanism that drives multiple age-related diseases, also causes osteoporosis. Recently, we systematically identified an accumulation of senescent cells, characterized by increased p16Ink4a and p21Cip1 levels and development of a senescence-associated secretory phenotype (SASP), in mouse bone/marrow and human bone with aging. We then demonstrated that elimination of senescent cells prevented age-related bone loss using multiple approaches, eg, treating old mice expressing a "suicide" transgene, INK-ATTAC, with AP20187 to induce apoptosis of p16Ink4a -senescent cells or periodically treating old wild-type mice with "senolytics," ie, drugs that eliminate senescent cells. Here, we investigate a possible role for estrogen in the regulation of cellular senescence using multiple approaches. First, sex steroid deficiency 2 months after ovariectomy (OVX, n = 15) or orchidectomy (ORCH, n = 15) versus sham surgery (SHAM, n = 15/sex) in young adult (4-month-old) wild-type mice did not alter senescence biomarkers or induce a SASP in bone. Next, in elderly postmenopausal women, 3 weeks of estrogen therapy (n = 10; 74 ± 5 years) compared with no treatment (n = 10; 78 ± 5 years) did not alter senescence biomarkers or the SASP in human bone biopsies. Finally, young adult (4-month-old) female INK-ATTAC mice were randomized (n = 17/group) to SHAM+Vehicle, OVX+Vehicle, or OVX+AP20187 for 2 months. As anticipated, OVX+Vehicle caused significant trabecular/cortical bone loss compared with SHAM+Vehicle. However, treatment with AP20187, which eliminates senescent cells in INK-ATTAC mice, did not rescue the OVX-induced bone loss or alter senescence biomarkers. Collectively, our data establish independent roles of estrogen deficiency and cellular senescence in the pathogenesis of osteoporosis, which has important implications for testing novel senolytics for skeletal efficacy, as these drugs will need to be evaluated in preclinical models of aging as opposed to the current FDA model of prevention of OVX-induced bone loss. © 2019 American Society for Bone and Mineral Research.

miR-219a-5p Regulates Rorß During Osteoblast Differentiation and in Age-related Bone Loss.

Developing novel approaches to treat skeletal disorders requires an understanding of how critical molecular factors regulate osteoblast differentiation and bone remodeling. We have reported that (1) retinoic acid receptor-related orphan receptor beta (Rorß) is upregulated in bone samples isolated from aged mice and humans in vivo; (2) Rorß expression is inhibited during osteoblastic differentiation in vitro; and (3) genetic deletion of Rorß in mice results in preservation of bone mass during aging. These data establish that Rorß inhibits osteogenesis and that strict control of Rorß expression is essential for bone homeostasis. Because microRNAs (miRNAs) are known to play important roles in the regulation of gene expression in bone, we explored whether a predicted subset of nine miRNAs regulates Rorß expression during both osteoblast differentiation and aging. Mouse osteoblastic cells were differentiated in vitro and assayed for Rorß and miRNA expression. As Rorß levels declined with differentiation, the expression of many of these miRNAs, including miR-219a-5p, was increased. We further demonstrated that miR-219a-5p was decreased in bone samples from old (24-month) mice, as compared with young (6-month) mice, concomitant with increased Rorß expression. Importantly, we also found that miR-219a-5p expression was decreased in aged human bone biopsies compared with young controls, demonstrating that this phenomenon also occurs in aging bone in humans. Inhibition of miR-219a-5p in mouse calvarial osteoblasts led to increased Rorß expression and decreased alkaline phosphatase expression and activity, whereas a miR-219a-5p mimic decreased Rorß expression and increased osteogenic activity. Finally, we demonstrated that miR-219a-5p physically interacts with Rorß mRNA in osteoblasts, defining Rorß as a true molecular target of miR-219a-5p. Overall, our findings demonstrate that miR-219a-5p is involved in the regulation of Rorß in both mouse and human bone. © 2018 American Society for Bone and Mineral Research.

Sympathetic ß1-adrenergic signaling contributes to regulation of human bone metabolism.

BACKGROUND: Evidence from rodent studies indicates that the sympathetic nervous system (SNS) regulates bone metabolism, principally via ß2-adrenergic receptors (ß2-ARs). Given the conflicting human data, we used multiple approaches to evaluate the role of the SNS in regulating human bone metabolism. METHODS: Bone biopsies were obtained from 19 young and 19 elderly women for assessment of ADRB1, ADRB2, and ADRB3 mRNA expression. We examined the relationship of ß-blocker use to bone microarchitecture by high-resolution peripheral quantitative CT in a population sample of 248 subjects. A total of 155 postmenopausal women were randomized to 1 of 5 treatment groups for 20 weeks: placebo; propranolol, 20 mg b.i.d.; propranolol, 40 mg b.i.d.; atenolol, 50 mg/day; or nebivolol, 5 mg/day. We took advantage of the ß1-AR selectivity gradient of these drugs (propranolol [nonselective] << atenolol [relatively ß1-AR selective] < nebivolol [highly ß1-AR selective]) to define the ß-AR selectivity for SNS effects on bone. RESULTS: ADRB1 and ADRB2, but not ADRB3, were expressed in human bone; patients treated clinically with ß1-AR-selective blockers had better bone microarchitecture than did nonusers, and relative to placebo, atenolol and nebivolol, but not propranolol, reduced the bone resorption marker serum C-telopeptide of type I collagen (by 19.5% and 20.6%, respectively; P < 0.01) and increased bone mineral density of the ultradistal radius (by 3.6% and 2.9%; P < 0.01 and P < 0.05, respectively). CONCLUSIONS: These 3 independent lines of evidence strongly support a role for adrenergic signaling in the regulation of bone metabolism in humans, principally via ß1-ARs. TRIAL REGISTRATION: ClinicalTrials.gov NCT02467400. FUNDING: This research was supported by the NIH (AG004875 and AR027065) and a Mayo Clinic Clinical and Translational Science Award (CTSA) (UL1 TR002377).

Osteoprotection Through the Deletion of the Transcription Factor Rorß in Mice.

There is a clinical need to identify new molecular targets for the treatment of osteoporosis, particularly those that simultaneously inhibit bone resorption while stimulating bone formation. We have previously shown in overexpression studies that retinoic acid receptor-related orphan receptor ß (Rorß) suppresses in vitro osteoblast differentiation. In addition, the expression of Rorß is markedly increased in bone marrow-derived mesenchymal stromal cells with aging in both mice and humans. Here we establish a critical role for Rorß in regulating bone metabolism using a combination of in vitro and in vivo studies. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing to demonstrate that loss of Rorß in osteoblasts enhances Wnt signaling, specifically through increased recruitment of ß-catenin to T-cell factor/lymphoid enhancer factor (Tcf/Lef) DNA binding sites in the promoters of the Wnt target genes Tcf7 and Opg. This resulted in increased osteogenic gene expression and suppressed osteoclast formation through increased osteoprotegerin (OPG) secretion in Rorß-deficient cells. Consistent with our in vitro data, genetic deletion of Rorß in both female and male mice resulted in preserved bone mass and microarchitecture with advancing age due to increased bone formation with a concomitant decrease in resorption. The improved skeletal phenotype in the Rorß-/- mice was also associated with increased bone protein levels of TCF7 and OPG. These data demonstrate that loss of Rorß has beneficial skeletal effects by increasing bone formation and decreasing bone resorption, at least in part through ß-catenin-dependent activation of the Wnt pathway. Thus, inhibition of Rorß represents a novel approach to potentially prevent or reverse osteoporosis. © 2017 American Society for Bone and Mineral Research.