Bone matrix quality in a developing high-fat diet mouse model is altered by RAGE deletion.

Overweightness and obesity in adolescents are epidemics linked to chronic low-grade inflammation and elevated fracture risk. The increased fracture risk observed in overweight/obese adolescence contrasts the traditional concept that high body mass is protective against fracture, and thus highlights the need to determine why weight gain becomes detrimental to fracture during growth and maturity. The Receptor for Advanced Glycation End products (RAGE) is a central inflammatory regulator that can influence bone metabolism. It remains unknown how RAGE removal impacts skeletal fragility in overweightness/obesity, and whether increased fracture risk in adolescents could result from low-grade inflammation deteriorating bone quality. We characterized the multiscale structural, mechanical, and chemical properties of tibiae extracted from adolescent C57BL/6J (WT) and RAGE null (KO) mice fed either low-fat (LF) or high-fat (HF) diet for 12 weeks starting at 6 weeks of age using micro-computed tomography, strength, Raman spectroscopy, and nanoindentation. Overweight/obese WT HF mice possessed degraded mineral-crystal quality and increased matrix glycoxidation in the form of pentosidine and carboxymethyl-lysine, with HF diet in females only showing reduced cortical surface expansion and TMD independently of RAGE ablation. Furthermore, in contrast to males, HF diet in females led to more material damage and plastic deformation. RAGE KO mitigated glycoxidative matrix accumulation, preserved mineral quantity, and led to increased E/H ratio in females. Taken together, these results highlight the complex, multi-scale and sex-dependent relationships between bone quality and function under overweightness, and identifies RAGE-controlled glycoxidation as a target to potentially preserve matrix quality and mechanical integrity.

High fat diet causes inferior vertebral structure and function without disc degeneration in RAGE-KO mice.

Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences. This study determined if high fat (HF) diet causes structural and functional changes to vertebrae and intervertebral discs (IVDs); if these changes are modulated in mice with systematic ablation for the receptor for advanced glycation endproducts (RAGE-KO); and if these changes are sex-dependent. Wild-type (WT) and RAGE-KO mice were fed a low fat (LF) or HF diet for 12 weeks starting at 6 weeks, representing the juvenile population. HF diet led to weight/fat gain, glucose intolerance, and increased cytokine levels (IL-5, MIG, and RANTES); with less fat gain in RAGE-KO females. Most importantly, HF diet reduced vertebral trabecular bone volume fraction and compressive and shear moduli, without a modifying effect of RAGE-KO, but with a more pronounced effect in females. HF diet caused reduced cortical area fraction only in WT males. Neither HF diet nor RAGE-KO affected IVD degeneration grade. Biomechanical properties of coccygeal motion segments were affected by RAGE-KO but not diet, with some interactions identified. In conclusion, HF diet resulted in inferior vertebral structure and function with some sex differences, no IVD degeneration, and few modifying effects of RAGE-KO. These structural and functional deficiencies with HF diet provide further evidence that diet can affect spinal structures and may increase the risk for spinal injury and degeneration with aging and additional stressors. Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences.

Leptin signaling and the intervertebral disc: Sex dependent effects of leptin receptor deficiency and Western diet on the spine in a type 2 diabetes mouse model.

Type 2 diabetes and obesity are associated with back pain in juveniles and adults and are implicated in intervertebral disc (IVD) degeneration. Hypercaloric Western diets are associated with both obesity and type 2 diabetes. The objective of this study was to determine if obesity and type 2 diabetes result in spinal pathology in a sex-specific manner using in vivo diabetic and dietary mouse models. Leptin is an appetite-regulating hormone, and its deficiency leads to polyphagia, resulting in obesity and diabetes. Leptin is also associated with IVD degeneration, and increased expression of its receptor was identified in degenerated IVDs. We used young, leptin receptor deficient (Db/Db) mice to mimic the effect of diet and diabetes on adolescents. Db/Db and Control mice were fed either Western or Control diets, and were sacrificed at 3 months of age. Db/Db mice were obese, while only female mice developed diabetes. Female Db/Db mice displayed altered IVD morphology, with increased intradiscal notochordal band area, suggesting delayed IVD cell proliferation and differentiation, rather than IVD degeneration. Motion segments from Db/Db mice exhibited increased failure risk with decreased torsional failure strength. Db/Db mice also had inferior bone quality, which was most prominent in females. We conclude that obesity and diabetes due to impaired leptin signaling contribute to pathological changes in vertebrae, as well as an immature IVD phenotype, particularly of females, suggesting a sex-dependent role of leptin in the spine.

Advanced glycation end products cause RAGE-dependent annulus fibrosus collagen disruption and loss identified using in situ second harmonic generation imaging in mice intervertebral disk in vivo and in organ culture models.

Aging and diabetes are associated with increased low-back pain and intervertebral disk (IVD) degeneration yet causal mechanisms remain uncertain. Advanced glycation end products (AGEs), which accumulate in IVDs from aging and are implicated in diabetes-related disorders, alter collagen and induce proinflammatory conditions. A need exists for methods that assess IVD collagen quality and degradation in order to better characterize specific structural changes in IVDs due to AGE accumulation and to identify roles for the receptor for AGEs (RAGE). We used multiphoton microscopy with second harmonic generation (SHG), collagen-hybridizing peptide (CHP), and image analysis methods to characterize effects of AGEs and RAGE on collagen quality and quantity in IVD annulus fibrosus (AF). First, we used SHG imaging on thin sections with an in vivo dietary mouse model and determined that high-AGE (H-AGE) diets increased AF fibril disruption and collagen degradation resulting in decreased total collagen content, suggesting an early degenerative cascade. Next, we used in situ SHG imaging with an ex vivo IVD organ culture model of AGE challenge on wild type and RAGE-knockout (RAGE-KO) mice and determined that early degenerative changes to collagen quality and degradation were RAGE dependent. We conclude that AGE accumulation leads to RAGE-dependent collagen disruption in the AF and can initiate molecular and tissue level collagen disruption. Furthermore, SHG and CHP analyzes were sensitive to collagenous alterations at multiple hierarchical levels due to AGE and may be useful in identifying additional contributors to collagen damage in IVD degeneration processes.

Elevated glycohemoglobin HbA1c is associated with low back pain in nonoverweight diabetics.

BACKGROUND CONTEXT: Low back pain (LBP) is a common complaint in clinical practice of multifactorial origin. Although obesity has been thought to contribute to LBP primarily by altering the distribution of mechanical loads on the spine, the additional contribution of obesity-related conditions such as diabetes mellitus (DM) to LBP has not been thoroughly examined. PURPOSE: To determine if there is a relationship between DM and LBP that is independent of body mass index (BMI) in a large cohort of adult survey participants. STUDY DESIGN: Retrospective analysis of prospectively collected National Health and Nutrition Examination Survey (NHANES) data to characterize associations between LBP, DM, and BMI in adults subdivided into 6 subpopulations: normal weight (BMI 18.5-25), overweight (BMI 25-30), and obese (BMI >30) diabetics and nondiabetics. Diabetes was defined with glycohemoglobin A1c (HbA1c) ≥6.5%. PATIENT SAMPLE: 11,756 participants from NHANES cohort. OUTCOME MEASURES: Percentage of LBP reported. METHODS: LBP reported in the 1999-2004 miscellaneous pain NHANES questionnaire was the dependent variable examined. Covariates included HbA1c, BMI, age, and family income ratio to poverty as continuous variables as well as race, gender, and smoking as binary variables. Individuals were further subdivided by weight class and diabetes status. Regression and graphical analyses were performed on the study population as a whole and also on subpopulations. RESULTS: Increasing HbA1c did not increase the odds of reporting LBP in the full cohort. However, multivariate logistic regression of the 6 subpopulations revealed that the odds of LBP significantly increased with increasing HbA1c levels in normal weight diabetics. No other subpopulations reported significant relationships between LBP and HbA1c. LBP was also significantly associated with BMI for normal weight diabetics and also for obese subjects regardless of their DM status. CONCLUSIONS: LBP is significantly related to DM status, but this relationship is complex and may interact with BMI. These results support the concept that LBP may be improved in normal weight diabetic subjects with improved glycemic control and weight loss, and that all obese LBP subjects may benefit from improved weight loss alone.

Dietary advanced glycation end-product consumption leads to mechanical stiffening of murine intervertebral discs.

Back pain is a leading cause of disability and is strongly associated with intervertebral disc (IVD) degeneration. Reducing structural disruption and catabolism in IVD degeneration remains an important clinical challenge. Pro-oxidant and structure-modifying advanced glycation end-products (AGEs) contribute to obesity and diabetes, which are associated with increased back pain, and accumulate in tissues due to hyperglycemia or ingestion of foods processed at high heat. Collagen-rich IVDs are particularly susceptible to AGE accumulation due to their slow metabolic rates, yet it is unclear whether dietary AGEs can cross the endplates to accumulate in IVDs. A dietary mouse model was used to test the hypothesis that chronic consumption of high AGE diets results in sex-specific IVD structural disruption and functional changes. High AGE diet resulted in AGE accumulation in IVDs and increased IVD compressive stiffness, torque range and failure torque, particularly for females. These biomechanical changes were likely caused by significantly increased AGE crosslinking in the annulus fibrosus, measured by multiphoton imaging. Increased collagen damage measured with collagen hybridizing peptide did not appear to influence biomechanical properties and may be a risk factor as these animals age. The greater influence of high AGE diet on females is an important area of future investigation that may involve AGE receptors known to interact with estrogen. We conclude that high AGE diets can be a source for IVD crosslinking and collagen damage known to be important in IVD degeneration. Dietary modifications and interventions that reduce AGEs warrant further investigation and may be particularly important for diabetics, in whom AGEs accumulate more rapidly.

Accumulation and localization of macrophage phenotypes with human intervertebral disc degeneration.

BACKGROUND CONTEXT: Chronic inflammation is an important component of intervertebral disc (IVD) degeneration, but there is limited knowledge about the identity and source of inflammatory cells involved with the degenerative processes. Macrophages can exhibit multiple phenotypes and are known inflammatory regulators in many tissues, but their phenotypes have not been characterized in IVD degeneration. PURPOSE: We aimed to characterize accumulation and localization of macrophages in IVD degeneration. STUDY DESIGN/SETTING: This is an exploratory study to characterize macrophage phenotypes in human cadaver IVDs and the effects of injury and degeneration using multiple immunohistochemistry methods. OUTCOME MEASURES: Percent positivity of immunohistochemical markers specific for CCR7, CD163, and CD206, and qualitative assessments of dual immunofluorescence and immunostaining localization were the outcome measures. METHODS: Macrophages were identified in human cadaveric IVDs with immunohistochemistry using cell surface markers CCR7, CD163, and CD206, which are associated with proinflammatory M1, remodeling M2c, and anti-inflammatory M2a phenotypes, respectively. Variations in the accumulation and localization of macrophage markers with degenerative grade across subjects and within donors are described. RESULTS: Cells expressing all three macrophage markers were found in all degenerative IVDs, but not in the healthiest IVDs. Cells expressing CCR7 and CD163, but not CD206, significantly increased with degenerative grade. Many cells also co-expressed multiple macrophage markers. Across all degenerative grades, CCR7+ and CD163+ were significantly more present in unhealthy nucleus pulposus (NP), annulus fibrosus (AF), and end plate (EP) regions exhibiting structural irregularities and defects. Positively stained cells in the NP and AF closely resembled resident IVD cells, suggesting that IVD cells can express macrophage cell surface markers. In the EP, there were increasing trends of positively stained cells with atypical morphology and distribution, suggesting a source for exogenous macrophage infiltration into the IVD. CONCLUSIONS: Chronic inflammatory conditions of IVD degeneration appear to involve macrophages or macrophage-like cells, as expression of multiple macrophage markers increased with degeneration, especially around unhealthy regions with defects and the EP. Knowledge of macrophage phenotypes and their localization better elucidates the complex injury and repair processes in IVDs and may eventually lead to novel treatments.

Mechanical signals protect stem cell lineage selection, preserving the bone and muscle phenotypes in obesity.

The incidence of obesity is rapidly rising, increasing morbidity and mortality rates worldwide. Associated comorbidities include type 2 diabetes, heart disease, fatty liver disease, and cancer. The impact of excess fat on musculoskeletal health is still unclear, although it is associated with increased fracture risk and a decline in muscular function. The complexity of obesity makes understanding the etiology of bone and muscle abnormalities difficult. Exercise is an effective and commonly prescribed nonpharmacological treatment option, but it can be difficult or unsafe for the frail, elderly, and morbidly obese. Exercise alternatives, such as low-intensity vibration (LIV), have potential for improving musculoskeletal health, particularly in conditions with excess fat. LIV has been shown to influence bone marrow mesenchymal stem cell differentiation toward higher-order tissues (i.e., bone) and away from fat. While the exact mechanisms are not fully understood, recent studies utilizing LIV both at the bench and in the clinic have demonstrated some efficacy. Here, we discuss the current literature investigating the effects of obesity on bone, muscle, and bone marrow and how exercise and LIV can be used as effective treatments for combating the negative effects in the presence of excess fat.

Diminished satellite cells and elevated adipogenic gene expression in muscle as caused by ovariectomy are averted by low-magnitude mechanical signals.

Age-related degeneration of the musculoskeletal system, accelerated by menopause, is further complicated by increased systemic and muscular adiposity. The purpose of this study was to identify at the molecular, cellular, and tissue levels the impact of ovariectomy on adiposity and satellite cell populations in mice and whether mechanical signals could influence any outcomes. Eight-week-old C57BL/6 mice were ovariectomized, with one half subjected to low-intensity vibration (LIV; 0.3 g/90 Hz, 15 min/day, 5 day/wk; n = 10) for 6 wk and the others sham vibrated (OVX; n = 10). Data are compared with age-matched, intact controls (AC; n = 10). In vivo µCT analysis showed that OVX mice gained 43% total (P < 0.001) and 125% visceral adiposity (P < 0.001) compared with their baseline after 6 wk, whereas LIV gained only 21% total (P = 0.01) and 70% visceral adiposity (P < 0.01). Relative to AC, expression of adipogenic genes (PPARγ, FABP4, PPARδ, and FoxO1) was upregulated in OVX muscle (P < 0.05), whereas LIV reduced these levels (P < 0.05). Adipogenic gene expression was inversely related to the percentage of total and reserve satellite cell populations in the muscle, with both declining in OVX compared with AC (-21 and -28%, respectively, P < 0.01). LIV mitigated these declines (-11 and -17%, respectively). These results provide further evidence of the negative consequences of estrogen depletion and demonstrate that mechanical signals have the potential to interrupt subsequent adipogenic gene expression and satellite cell suppression, emphasizing the importance of physical signals in protecting musculoskeletal integrity and slowing the fat phenotype.