Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?

Bacteriophages (phages) and their preys are engaged in an evolutionary arms race driving the co-adaptation of their attack and defense mechanisms. In this context, phages have evolved diverse anti-CRISPR proteins to evade the bacterial CRISPR-Cas immune system, and propagate. Anti-CRISPR proteins do not share much resemblance with each other and with proteins of known function, which raises intriguing questions particularly relating to their modes of action. In recent years, there have been many structure-function studies shedding light on different CRISPR-Cas inhibition strategies. As the anti-CRISPR field of research is rapidly growing, it is opportune to review the current knowledge on these proteins, with particular emphasis on the molecular strategies deployed to inactivate distinct steps of CRISPR-Cas immunity. Anti-CRISPR proteins can be orthosteric or allosteric inhibitors of CRISPR-Cas machineries, as well as enzymes that irreversibly modify CRISPR-Cas components. This repertoire of CRISPR-Cas inhibition mechanisms will likely expand in the future, providing fundamental knowledge on phage-bacteria interactions and offering great perspectives for the development of biotechnological tools to fine-tune CRISPR-Cas-based gene edition.

The relationships between bone variables and physical fitness across the BMI spectrum in young adult women.

In this cross-sectional study we aimed to evaluate the relationship between physical fitness and bone variables across the body mass index (BMI) spectrum in women aged 20-35 years. The study included 13 underweight women (BMI < 18.5 kg/m2), 24 normal weight women (BMI 18.5-24.9 kg/m2), and 20 overweight/obese women (BMI ≥ 25 kg/m2) aged between 20 and 35 years. Bone mineral density (BMD) and content (BMC) at the whole body, lumbar spine, and femoral neck, lumbar spine trabecular bone score, femoral neck geometry were assessed using dual-energy X-ray absorptiometry. Cardiorespiratory fitness and lower limb muscle power were estimated using the 20-m shuttle run test and the Sargent jump test, respectively. The associations between bone variables and physical fitness were different according to BMI categories. Correlations between physical fitness and bone parameters are particularly significant in normal BMI and less significant in low and high BMI. Multivariate ANCOVA regression models demonstrated that absolute VO2max (L/min) is a strong determinant of all the bone parameters regardless of BMI. Implementing strategies for increasing VO2max (L/min) by increasing lean mass and promoting resistance and/or high-intensity interval training could be effective to optimize bone health in underweight and overweight young adult women.

Bone Geometric Properties of the Femoral Neck in Underweight Eumenorrheic Women.

The aim of this study was to describe femoral neck (FN) geometry among eumenorrheic underweight women around the age of peak bone mass. Proximal femur geometry and body composition were assessed in 12 underweight women and in 24 healthy controls using dual-energy X-ray absorptiometry. The Hip Structural Analysis program was used to determine bone geometry at the FN. The cross-sectional area (CSA) and the cross-sectional moment of inertia (CSMI) were significantly lower in underweight women than in controls (p < 0.05). There was a trend toward lower sectional modulus (Z) and strength index in underweight women (p < 0.15). Body weight, body mass index, and lean mass (LM) were positively correlated with CSA, CSMI, Z, and neck-shaft angle (r = 0.428-0.611, p < 0.05). After controlling for body weight, body mass index, and LM, the differences in CSA, CSMI, Z, and neck-shaft angle were no more statistically significant between the 2 groups. The multivariate analysis retained LM as the main predictor of CSA, CSMI, and Z in the whole population. The present study suggests that thinness is associated with low resistance to axial forces (CSA) and bending load (Z and CSMI) in adult eumenorrheic women. LM seems to be a key determinant of FN geometry in underweight women.

Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding.

Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b λx isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of α-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form.

Assessment of the zonal variation of perfusion parameters in the femoral head: a 3-T dynamic contrast-enhanced MRI pilot study.

OBJECTIVE: The objective was to describe MR perfusion characteristics of the femoral head, with a focus on the subchondral bone. MATERIALS AND METHODS: This prospective monocentric study was approved by our local Ethics Committee. Written informed consent was obtained from all subjects. Dynamic contrast-enhanced MRI of the right hip was performed in 59 adults with suspected spondyloarthritis (32 women, 28 men). Mean age was 37.5 (±12.5) years. Regions of interest were drawn in the femoral head epiphysis, in the subchondral areas the most exposed to mechanical load (superolateral, anterosuperior, and posterior zones) and in areas less exposed to mechanical load (inferior subchondral zone and center of the femoral head). Semi-quantitative and pharmacokinetic parameters were calculated using the Tofts model. Statistical analysis was performed with a linear mixed model to compare the perfusion parameters in the different femoral head zones. RESULTS: Extravascular extracellular volume and area under the curve were lower in the superolateral zone than in the inferior zone (p = 0.0135 and p < 0.0001 respectively) and the central zone (p = 0.007 and p = 0.0134 respectively). Extravascular extracellular volume and rate constant were lower in the anterosuperior zone than in the inferior zones (p = 0.011 and p = 0.029). In the anterosuperior zone, extravascular extracellular volume was lower, and time to peak was higher than in the central zones (p = 0.0056 and p = 0.0013 respectively). No significant differences were found for any values between other paired zones. CONCLUSION: The perfusion of femoral head subchondral bone assessed with dynamic contrast-enhanced magnetic resonance imaging is not homogeneous: the areas exposed to more mechanical loading are less perfused.

Long-Term Energy Deficit in Mice Causes Long-Lasting Hypothalamic Alterations after Recovery.

Although the short-term effects of fasting or energy deficit on hypothalamic neuropeptide circuitries are now better understood, the effects of long-term energy deficit and refeeding remain to be elucidated. We showed that after a long-term energy deficit, mice exhibited persistent hypoleptinemia following the refeeding period despite restoration of fat mass, ovarian activity, and feeding behavior. We aimed to examine the hypothalamic adaptations after 10 weeks of energy deficit and after 10 further weeks of nutritional recovery. To do so, we assessed the mRNA levels of the leptin receptor and the main orexigenic and anorexigenic peptides, and their receptors regulated by leptin. Markers of hypothalamic inflammation were assessed as leptin can also participate in this phenomenon. Long-term time-restricted feeding and separation induced significant increase in mRNA levels of hypothalamic orexigenic peptides, while both Y1 and Y5 receptor mRNAs were downregulated. No changes occurred in the mRNA levels of orexin (OX), melanin-concentrating hormone, pro-opiomelanocortin, 26RFa (26-amino acid RF-amide peptide), and their receptors despite an increase in the expression of melanocortin receptors (MC3-R and MC4-R) and OXR1 (OX receptor 1). The refeeding period induced an overexpression of leptin receptor mRNA in the hypothalamus. The other assessed mRNA levels were normalized except for Y2, Y5, MC3-R, and MC4-R, which remained upregulated. No convincing changes were observed in neuroinflammatory markers, even if interleukin-1ß mRNA levels were increased in parallel with those of Iba1 (ionized calcium-binding adaptor molecule 1), a marker of microglial activation. Normalization of leptin-regulated functions and hypothalamic gene expressions in refed mice with low plasma leptin levels could be sustained by recalibration of hypothalamic sensitivity to leptin.

Bone Samples Extracted from Embalmed Subjects Are Not Appropriate for the Assessment of Bone Quality at the Molecular Level Using Raman Spectroscopy.

Bone samples extracted from embalmed cadavers are commonly used as controls in the study of bone. The effects of embalmment on the molecular composition of bone are unknown. The objective of this study was to determine the effect of embalmment on the molecular composition and structure of bone, as evaluated by Raman spectroscopy. Bone samples of femoral heads from five embalmed donors and five fresh-frozen donors were compared using Raman microspectroscopy with DuoScan technology. Physicochemical parameters simultaneously describing the organic and mineral phases of bone were compared using the Mann-Whitney U test. Partial least squares discriminant analysis (PLS-DA) was used to determine specific Raman spectral features of each group. Study of the mineral phase showed a 15% reduction of the mineral-to-matrix ratio (p < 0.001), an 8% decrease of type B carbonate substitution (p < 0.001), and a 2% increase in crystallinity (p < 0.001) in the embalmed donors group compared to those of the fresh donors group. Regarding the organic phase of bone, the hydroxyproline-to-proline ratio was increased by 18% in the embalmed group (p < 0.001), with no variation in both the relative proteoglycan content (GAG/CH3) (p = 0.08) and collagen maturity (p = 0.57). PLS-DA showed that the embalmed group was characterized mainly by peaks assigned to hydroxyproline, lipids, and collagen. Embalmment induces significant modifications of the molecular composition of bone. Bone samples from embalmed subjects should be avoided as controls for Raman spectroscopy studies. Preservation procedures performed prior to bone sampling should be reported in studies using human cadaver samples.

Adipogenic RNAs are transferred in osteoblasts via bone marrow adipocytes-derived extracellular vesicles (EVs).

BACKGROUND: In osteoporosis, bone loss is accompanied by increased marrow adiposity. Given their proximity in the bone marrow and their shared origin, a dialogue between adipocytes and osteoblasts could be a factor in the competition between human Mesenchymal Stem Cells (hMSC) differentiation routes, leading to adipocyte differentiation at the expense of osteoblast differentiation. The adipocyte/osteoblast balance is highly regulated at the level of gene transcription. In our work, we focused on PPARgamma, CEBPalpha and CEBPdelta, as these transcription factors are seen as master regulators of adipogenesis and expressed precociously, and on leptin and adiponectin, considered as adipocyte marker genes. In 2010, our group has demonstrated, thanks to a coculture model, that in the presence of hMSC-derived adipocytes (hMSC-Adi), hMSC-derived osteoblasts (hMSC-Ost) express lesser amounts of osteogenic markers but exhibit the expression of typical adipogenic genes. Nevertheless, the mechanisms underlying this modulation of gene expression are not clarified. Recently, adipocytes were described as releasing extracellular vesicles (EVs), containing and transferring adipocyte specific transcripts, like PPARgamma, leptin and adiponectin. Here, we investigated whether EVs could be the way in which adipocytes transfer adipogenic RNAs in our coculture model. RESULTS: We observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. Moreover, EVs were isolated from conditioned media collected from cultures of hMSC at different stages of adipocyte differentiation and these specific adipogenic transcripts were detected inside. Finally, thanks to interspecies conditioned media exposition, we could highlight for the first time a horizontal transfer of adipogenic transcripts from medullary adipocytes to osteoblasts. CONCLUSIONS: Here, we have shown, for the first time, RNA transfer between hMSC-derived adipocytes and osteoblasts through EVs. Additional studies are needed to clarify if this mechanism has a role in the adipocytic switch driven on osteoblasts by adipocytes inside bone marrow and if EVs could be a target component to regulate the competition between osteoblasts and adipocytes in the prevention or in the therapy of osteoporosis and other osteopenia.

Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation.

BACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipogenic pathway. Supporting this hypothesis the competition between adipogenic and osteogenic lineages was widely demonstrated on partially homogeneous cell populations. However, some data from mouse models showed the existence of an independent relationship between bone mineral content and bone marrow adiposity. Therefore, the combination of adipogenesis and osteogenesis in primary culture would be helpful to determine if this competition would be observed on a whole bone marrow stromal cell population in a culture medium allowing both lineages. In this aim, mouse bone marrow stromal cells were cultured in a standard osteogenic medium added with different concentrations of Dexamethasone, known to be an important regulator of mesenchymal progenitor cell differentiation. RESULTS: Gene expression of osteoblast and adipocyte markers, biochemical and physical analyses demonstrated the presence of both cell types when Dexamethasone was used at 100 nM. Overall, our data showed that in this co-differentiation medium both differentiation lineages were enhanced compared to classical adipogenic or osteogenic culture medium. This suggests that in this model, adipocyte phenotype does not seem to increase at the expense of the osteoblast lineage. CONCLUSION: This model appears to be a promising tool to study osteoblast and adipocyte differentiation capabilities and the interactions between these two processes.

Variations of secretome profiles according to conditioned medium preparation: The example of human mesenchymal stem cell-derived adipocytes.

One challenging point in analyzing cellular secretome collected as conditioned medium is cross-contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum-free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum-free adipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and 1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation.

3 Tesla (1) H MR spectroscopy of hip bone marrow in a healthy population, assessment of normal fat content values and influence of age and sex.

PURPOSE: To evaluate in a healthy population normal spectroscopic fat content (FC) values of the hip bone marrow and to assess the influence of age and sex on bone marrow conversion. MATERIALS AND METHODS: Eighty volunteers (40 men; 40 women; ages: 20-60 years; divided into four consecutive groups) underwent acetabulum, femoral head, femoral neck, greater trochanter, and diaphysis localized (1) H MR spectroscopy. FC values of each anatomical site were obtained according to the following formula: Fat content = CH2 /(CH2 + Water)*100. To assess bone marrow conversion, a spectroscopic conversion index (SCI) was calculated as FC neck/FC greater trochanter. RESULTS: FC values showed a gradient as follows: greater trochanter > femoral head > femoral neck > diaphysis > acetabulum in every age group both in men and in women. SCI increased with age both in men and women, showing lower values in women for every age group. CONCLUSION: We obtained normal spectroscopic FC values from different areas of the hip, according to age and sex. These values may be used as reference values to evaluate, by the means of (1) H MR spectroscopy, pathological conditions affecting hip bone marrow.

RNA Secondary Structure Modeling Following the IPANEMAP Workflow.

The structure of RNA molecules and their complexes are crucial for understanding biology at the molecular level. Resolving these structures holds the key to understanding their manifold structure-mediated functions ranging from regulating gene expression to catalyzing biochemical processes. Predicting RNA secondary structure is a prerequisite and a key step to accurately model their three dimensional structure. Although dedicated modelling software are making fast and significant progresses, predicting an accurate secondary structure from the sequence remains a challenge. Their performance can be significantly improved by the incorporation of experimental RNA structure probing data. Many different chemical and enzymatic probes have been developed; however, only one set of quantitative data can be incorporated as constraints for computer-assisted modelling. IPANEMAP is a recent workflow based on RNAfold that can take into account several quantitative or qualitative data sets to model RNA secondary structure. This chapter details the methods for popular chemical probing (DMS, CMCT, SHAPE-CE, and SHAPE-Map) and the subsequent analysis and structure prediction using IPANEMAP.

Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells.

Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exert their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.

Structural Insights into the Dimeric Form of Bacillus subtilis RNase Y Using NMR and AlphaFold.

RNase Y is a crucial component of genetic translation, acting as the key enzyme initiating mRNA decay in many Gram-positive bacteria. The N-terminal domain of Bacillus subtilis RNase Y (Nter-BsRNaseY) is thought to interact with various protein partners within a degradosome complex. Bioinformatics and biophysical analysis have previously shown that Nter-BsRNaseY, which is in equilibrium between a monomeric and a dimeric form, displays an elongated fold with a high content of α-helices. Using multidimensional heteronuclear NMR and AlphaFold models, here, we show that the Nter-BsRNaseY dimer is constituted of a long N-terminal parallel coiled-coil structure, linked by a turn to a C-terminal region composed of helices that display either a straight or bent conformation. The structural organization of the N-terminal domain is maintained within the AlphaFold model of the full-length RNase Y, with the turn allowing flexibility between the N- and C-terminal domains. The catalytic domain is globular, with two helices linking the KH and HD modules, followed by the C-terminal region. This latter region, with no function assigned up to now, is most likely involved in the dimerization of B. subtilis RNase Y together with the N-terminal coiled-coil structure.

Cell-specific effects of TNF-α and IL-1ß on alkaline phosphatase: implication for syndesmophyte formation and vascular calcification.

Tumor necrosis factor (TNF)-α and interleukin (IL)-1ß stimulate tissue non-specific alkaline phosphatase (TNAP) activity and mineralization in cultures of vascular smooth muscle cells (VSMCs). They are, therefore, considered as stimulators of vascular calcification in the context of atherosclerosis and diabetes type 2. In contrast, although ankylosing spondylitis (AS) leads to the formation of syndesmophytes, which are ectopic ossifications from entheses (where ligaments, tendons and capsules are attached to bone), anti-TNF-α therapies fail to block bone formation in this disease. In this context, our aims were to compare the effects of TNF-α and IL-1ß on TNAP activity and mineralization in entheseal cells and VSMCs. Organotypic cultures of mouse ankle entheses were treated or not with TNF-α and IL-1ß for 5 days. Micro-computed tomography was performed to determine trabecular bone parameters, and histology to assess TNAP activity and mineralization. Human mesenchymal stem cells cultured in pellets in chondrogenic conditions and human VSMCs were also used to determine the effects of cytokines on TNAP activity and expression, measured by quantitative PCR. In organotypic cultures, TNF-α and IL-1ß significantly reduced the tibia BV/TV ratio. They also inhibited TNAP activity in entheseal chondrocytes in situ, and in mouse and human chondrocytes in vitro. In contrast, TNF-α stimulated TNAP expression and activity in human VSMCs. These differences were likely due to cell-specific effects of peroxisome proliferator-activated receptor γ (PPARγ), which is inhibited by TNF-α. Indeed, in human chondrocytes and VSMCs, the PPARγ inhibitor GW-9662 displayed the same opposite effects as TNF-α on TNAP expression. In conclusion, whereas TNF-α and IL-1ß stimulate TNAP activity in VSMCs, they inhibit it in entheseal cells in situ and on chondrocytes in vitro. The identification of PPARγ as a likely mediator of cytokine effects deserves consideration for future research on the mechanisms of ectopic ossification.

Human osteoblasts derived from mesenchymal stem cells express adipogenic markers upon coculture with bone marrow adipocytes.

In osteoporosis, bone loss is accompanied by greater adiposity in the marrow. Given the cellular proximity within the bone marrow, we wondered whether adipocytes might have a paracrine impact on osteoblast differentiation. To test this hypothesis, we cocultured adipocytes with osteoblasts derived from mesenchymal stem cells (MSCs) in the absence of direct cell contact and then analyzed gene expression changes in the osteoblastic population by using real-time reverse transcription polymerase chain reaction. We found that, upon coculture, MSC-derived osteoblasts showed appearance of adipogenic (lipoprotein lipase, leptin) and decrease of osteogenic (osteocalcin) mRNA markers. Our results indicate that in vitro, MSC-derived adipocytes are capable of inducing MSC-derived osteoblasts to differentiate to an adipocyte phenotype. These new data suggest that (i) transdifferentiation of committed osteoblasts into adipocytes may contribute to the increase in marrow fat content at the expense of bone-forming cells and (ii) this switch might be initiated by the adipocytes themselves.

Adipocyte-induced transdifferentiation of osteoblasts and its potential role in age-related bone loss.

Our preliminary findings have lead us to propose bone marrow adipocyte secretions as new contributors to bone loss. Indeed, using a coculture model based on human bone marrow stromal cells, we previously showed that soluble factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. In this study, microarray gene expression profiling showed profound transcriptomic changes in osteoblasts following coculture and confirmed the enrichment of the adipocyte gene signature. Double immunofluorescence microscopic analyses demonstrated the coexpression of adipogenic and osteoblastic specific markers in individual cells, providing evidence for a transdifferentiation event. At the molecular level, this conversion was associated with upregulated expression levels of reprogramming genes and a decrease in the DNA methylation level. In line with these in vitro results, preliminary immunohistochemical analysis of bone sections revealed adipogenic marker expression in osteoblasts from elderly subjects. Altogether, these data suggest that osteoblast transdifferentiation could contribute to decreased bone mass upon ageing.

Sirtuin 1 deficiency decreases bone mass and increases bone marrow adiposity in a mouse model of chronic energy deficiency.

Sirtuin of type 1 (Sirt1), a class III HDAC, is known to be involved in the regulation of differentiation of skeletal stem cells (SSCs) into osteoblasts and adipocytes. In caloric restriction, it has been shown that the expression and activity of Sirt1 is a tissue-dependent regulation. However, at present, no study has focused on the link between Sirt1, bone marrow adiposity (BMA) and osteoporosis related to anorexia nervosa (AN). Thus, the aims of this work were to (i) determine BMA and bone changes in a mouse model replicating the phenotypes of AN (separation-based anorexia model (SBA)); (ii) determine the expression of Sirt1 in bone marrow stromal cells (BMSCs) extracted from these mice and identify their differentiation capacities; (iii) study the effects of pharmacological activation and inhibition of Sirt1 on the osteoblastogenesis and adipogenesis of these cells and (iiii) delineate the molecular mechanism by which Sirt1 could regulate osteogenesis in an SBA model. Our results demonstrated that SBA protocol induces an increase in BMA and alteration of bone architecture. In addition, BMSCs from restricted mice present a down-regulation of Sirt1 which is accompanied by an increase in adipogenesis at expense of osteogenesis. After a 10-day organotypic culture, tibias from SBA mice displayed low levels of Sirt1 mRNA which are restored by resveratrol treatment. Interestingly, this recovery of Sirt1 levels also returned the BMA, BV/TV and Tb.Th in cultured tibias from SBA mice to normal levels. In contrast of down-regulation of Sirt1 expression induced by sirtinol treatment, stimulation of Sirt1 expression by resveratrol lead to a decrease in adipogenesis and increase in osteogenesis. Finally, to investigate the molecular mechanisms by which Sirt1 could regulate osteogenesis in the SBA model, the acetylation levels of Runx2 and Foxo1 transcription factors were determined. Our data show that this chronic energy deficiency in female mice causes a decrease in BMSC activity, resulting in critical changes to Runx2 and Foxo1 acetylation levels and thus to their activity. Altogether, these data suggest that Sirt1 could be considered as a potential therapeutic target in osteoporosis related to AN.

Marrow adiposity and bone: Review of clinical implications.

There is growing interest in the relationship between bone marrow fat (BMF) and skeletal health. Progress in clinical studies of BMF and skeletal health has been greatly enhanced by recent technical advances in our ability to measure BMF non-invasively. Magnetic resonance imagery (MRI) with or without spectroscopy is currently the standard technique for evaluating BMF content and composition in humans. This review focuses on clinical studies of marrow fat and its relationship with bone. The amount of marrow fat is associated with bone mineral density (BMD). Several studies have reported a significant negative association between marrow fat content and BMD in both healthy and osteoporotic populations. There may also be a relationship between marrow fat and fracture (mostly vertebral fracture), but data are scarce and further studies are needed. Furthermore, a few studies suggest that a lower proportion of unsaturated lipids in vertebral BMF may be associated with reduced BMD and greater prevalence of fracture. Marrow fat might be influenced by metabolic diseases associated with bone loss and fractures, such as diabetes mellitus, obesity and anorexia nervosa. An intriguing aspect of bariatric (weight loss) surgery is that it induces bone loss and fractures, but with different impacts on marrow fat depending on diabetic status. In daily practice, the usefulness for clinicians of assessing marrow fat using MRI is still limited. However, the perspectives are exciting, particularly in terms of improving the diagnosis and management of osteoporosis. Further studies are needed to better understand the regulators involved in the marrow fat-bone relationship and the links between marrow fat, other fat depots and energy metabolism.

Adipokines and bone status in a cohort of anorexic patients.

INTRODUCTION: Bone loss in anorexia nervosa (AN) is multifactorial; its mechanisms are not yet clearly understood and may vary depending on disease duration and severity. To determine to what extent adipokines may be involved in the bone alterations found in anorexic patients, we evaluated plasma levels for leptin, adiponectin and Pref-1 against other clinical and biological parameters in a population of anorexic patients split according to weight and bone status. METHODS: Plasma concentrations of leptin, total adiponectin, high molecular weight (HMW) adiponectin, and Pref-1 were measured. The ratio of HMW adiponectin to total adiponectin - HMW (percentage) - was calculated. We divided our population into 5 groups with different phenotypes characterizing the severity of the disease and/or the severity of bone involvement: 1 - Normal BMD and body mass index (BMI): recovery from AN; 2 - Osteopenia (-217kg/m2; 3 - Osteopenia and BMI≤17kg/m2; 4 - Osteoporosis (Z-score≤-2) and BMI>17kg/m2; 5 - Osteoporosis and BMI≤17kg/m2. RESULTS: The study involved 80 anorexia nervosa patients. Mean BMI was 16.8±2.4kg/m2. No significant difference was found in total and HMW adiponectin plasma concentrations between the 5 groups. HMW (percentage) was significantly higher in group 5 compared to group 1. Leptin was significantly lower in groups 3 and 5 compared to the other groups. For the whole group femoral neck and hip BMD correlated negatively with total adiponectin and HMW adiponectin. No correlation was found between BMD (whatever the site) and plasma leptin. Multivariate analysis revealed that 2 factors - leptin and BMI - explained 10% of the variance in spine BMD. For femoral neck BMD, the 2 explanatory factors were BMI and total adiponectin which explained 14% of the variance in BMD. For total hip BMD, 27% of the variance in BMD was explained by 3 factors: leptin, BMI, and total adiponectin. CONCLUSION: Bone status in anorexia nervosa is mainly determined by BMI, leptin and adiponectin.