Julie Lacombe

Osteocalcin regulates murine and human fertility through a pancreas-bone-testis axis

The osteoblast-derived hormone osteocalcin promotes testosterone biosynthesis in the mouse testis by binding to GPRC6A in Leydig cells. Interestingly, Osteocalcin-deficient mice exhibit increased levels of luteinizing hormone (LH), a pituitary hormone that regulates sex steroid synthesis in the testes. These observations raise the question of whether LH regulates osteocalcins reproductive effects. Additionally, there is growing evidence that osteocalcin levels are a reliable marker of insulin secretion and sensitivity and circulating levels of testosterone in humans, but the endocrine function of osteocalcin is unclear. Using mouse models, we found that osteocalcin and LH act in 2 parallel pathways and that osteocalcin-stimulated testosterone synthesis is positively regulated by bone resorption and insulin signaling in osteoblasts. To determine the importance of osteocalcin in humans, we analyzed a cohort of patients with primary testicular failure and identified 2 individuals harboring the same heterozygous missense variant in one of the transmembrane domains of GPRC6A, which prevented the receptor from localizing to the cell membrane. This study uncovers the existence of a second endocrine axis that is necessary for optimal male fertility in the mouse and suggests that osteocalcin modulates reproductive function in humans.

Acute Myelogenous Leukemia-Induced Sympathetic Neuropathy Promotes Malignancy in an Altered Hematopoietic Stem Cell Niche

Perivascular mesenchymal stem and progenitor cells (MSPCs) are critical for forming a healthy hematopoietic stem cell (HSC) niche. However, the interactions and influence of acute myelogenous leukemia (AML) stem cells with the microenvironment remain largely unexplored. We have unexpectedly found that neuropathy of the sympathetic nervous system (SNS) promotes leukemic bone marrow infiltration in an MLL-AF9 AML model. Development of AML disrupts SNS nerves and the quiescence of Nestin(+) niche cells, leading to an expansion of phenotypic MSPCs primed for osteoblastic differentiation at the expense of HSC-maintaining NG2(+) periarteriolar niche cells. Adrenergic signaling promoting leukemogenesis is transduced by the β2, but not β3, adrenergic receptor expressed on stromal cells of leukemic bone marrow. These results indicate that sympathetic neuropathy may represent a mechanism for the malignancy in order to co-opt the microenvironment and suggest separate mesenchymal niche activities for malignant and healthy hematopoietic stem cells in the bone marrow.

Regulation of energy metabolism by the skeleton: osteocalcin and beyond.

The skeleton has recently emerged as an endocrine organ implicated in the regulation of glucose and energy metabolism. This function of bone is mediated, at least in part, by osteocalcin, an osteoblast-derived protein acting as a hormone stimulating insulin sensitivity, insulin secretion and energy expenditure. Osteocalcin secretion and bioactivity is in turn regulated by several hormonal cues including insulin, leptin, the sympathetic nervous system and glucocorticoids. Recent findings support the notion that osteocalcin functions and regulations are conserved between mice and humans. Moreover, studies in mice suggest that osteocalcin could represent a viable therapeutic approach for the treatment of obesity and insulin resistance. In this review, we summarize the current knowledge on osteocalcin functions, its various modes of action and the mechanisms implicated in the control of this hormone.

Scl regulates the quiescence and the long-term competence of hematopoietic stem cells

The majority of long-term reconstituting hematopoietic stem cells (LT-HSCs) in the adult is in G(0), whereas a large proportion of progenitors are more cycling. We show here that the SCL/TAL1 transcription factor is highly expressed in LT-HSCs compared with short-term reconstituting HSCs and progenitors and that SCL negatively regulates the G(0)-G(1) transit of LT-HSCs. Furthermore, when SCL protein levels are decreased by gene targeting or by RNA interference, the reconstitution potential of HSCs is impaired in several transplantation assays. First, the mean stem cell activity of HSCs transplanted at approximately 1 competitive repopulating unit was 2-fold decreased when Scl gene dosage was decreased. Second, Scl(+/-) HSCs were at a marked competitive disadvantage with Scl(+/+) cells when transplanted at 4 competitive repopulating units equivalent. Third, reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was decreased compared with controls. At the molecular level, we found that SCL occupies the Cdkn1a and Id1 loci in primary hematopoietic cells and that the expression levels of these 2 regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G(0)-G(1) transition in HSCs and regulates their long-term competence.

A RANKL–PKCβ–TFEB signaling cascade is necessary for lysosomal biogenesis in osteoclasts

Bone resorption by osteoclasts requires a large number of lysosomes that release proteases in the resorption lacuna. Whether lysosomal biogenesis is a consequence of the action of transcriptional regulators of osteoclast differentiation or is under the control of a different and specific transcriptional pathway remains unknown. We show here, through cell-based assays and cell-specific gene deletion experiments in mice, that the osteoclast differentiation factor RANKL promotes lysosomal biogenesis once osteoclasts are differentiated through the selective activation of TFEB, a member of the MITF/TFE family of transcription factors. This occurs following PKCβ phosphorylation of TFEB on three serine residues located in its last 15 amino acids. This post-translational modification stabilizes and increases the activity of this transcription factor. Supporting these biochemical observations, mice lacking in osteoclasts--either TFEB or PKCβ--show decreased lysosomal gene expression and increased bone mass. Altogether, these results uncover a RANKL-dependent signaling pathway taking place in differentiated osteoclasts and culminating in the activation of TFEB to enhance lysosomal biogenesis-a necessary step for proper bone resorption.

In vivo analysis of the contribution of bone resorption to the control of glucose metabolism in mice.

Osteocalcin is a hormone produced in bones by osteoblasts and regulating energy metabolism. While osteocalcin exists in two forms, γ-carboxylated and undercarboxylated only the latter appears to function as a hormone in vivo. It has been proposed recently that osteoclasts, the bone-resorbing cells, are responsible of decarboxylating, i.e. activating osteocalcin. To address the role of osteoclasts in the maintenance of energy metabolism we analyzed mutant mouse strains harboring either an increase or a decrease in osteoclasts number. Osteoprotegerin-deficient mice that are characterized by an increase in the number of osteoclasts demonstrate an increase in serum levels of undercarboxylated osteocalcin and are significantly more glucose tolerant than WT animals. Conversely, osteoclasts ablation in mice results in a decrease in serum undercarboxylated osteocalcin levels and in reduced glucose tolerance. These results support the notion that osteoclasts are controlling glucose metabolism at least in part through the regulation of osteocalcin decarboxylation.

Phenotypic Characterization of MIP-CreERT1Lphi Mice With Transgene-Driven Islet Expression of Human Growth Hormone

There is growing concern over confounding artifacts associated with β-cell–specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible β-cell–specific transgenic (MIP-CreERT1Lphi) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in α-cell number and serum glucagon were significantly blunted in MIP-CreERT1Lphi mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for β-cell research.

Pessary use in advanced pelvic organ prolapse

The objective of this study was to review our experience with pessary use for advanced pelvic organ prolapse. Charts of patients treated for Stage III and IV prolapse were reviewed. Comparisons were made between patients who tried or refused pessary use. A successful trial of pessary was defined by continued use; a failed trial was defined by a patient’s discontinued use. Thirty-two patients tried a pessary; 45 refused. Patients who refused a pessary were younger, had lesser degree of prolapse, and more often had urinary incontinence. Most patients (62.5%) continued pessary use and avoided surgery. Unsuccessful trial of pessary resorting to surgery included four patients (33%) with unwillingness to maintain, three patients (25%) with inability to retain and two patients (17%) with vaginal erosion and/or discharge. Our findings suggest that pessary use is an acceptable first-line option for treatment of advanced pelvic organ prolapse.

GGCX and VKORC1 inhibit osteocalcin endocrine functions.

Cell-specific gene inactivation experiments delineate the functions of the enzymes required for osteocalcin modification and demonstrate that it is its uncarboxylated form that acts as a hormone.

ORIGINAL RESEARCH—WOMEN’S SEXUAL HEALTH: Genital Sensation and Sexual Function in Women Bicyclists and Runners: Are Your Feet Safer than Your Seat?

INTRODUCTION Bicycling is associated with neurological impairment and impotence in men. Similar deficits have not been confirmed in women. AIM To evaluate the effects of bicycling on genital sensation and sexual function in women. METHODS Healthy, premenopausal, competitive women bicyclists and runners (controls) were compared. MAIN OUTCOME MEASURES (1) Genital vibratory thresholds (VTs) were determined using the Medoc Vibratory Sensation Analyzer 3000. (2) Sexual function and sexually related distress were assessed by the Dennerstein Personal Experience Questionnaire (SPEQ) and the Female Sexual Distress Scale (FSDS). RESULTS Forty-eight bicyclists and 22 controls were enrolled. The median age was 33 years. The bicyclists were older, had higher body mass indices (BMIs), were more diverse in their sexual orientation, and were more likely to have a current partner. Bicyclists rode an average of 28.3 +/- 19.7 miles/day (range 4-100), 3.8 +/- 1.5 days/week, for an average of 2.1 +/- 1.8 hours/ride. The mean number of years riding was 7.9 +/- 7.1 years (range 0.5-30). Controls ran an average of 4.65 +/- 2.1 miles/day (range 1.5-8) and 5.0 +/- 1.2 days/week. On bivariate analysis, bicyclists had significantly higher VTs than runners, indicating worse neurological function at all sites (P < 0.05). Multivariate analysis found significant correlations between higher VTs and bicycling at the left and right perineum, posterior vagina, left and right labia. Increasing VTs at the clitoris, anterior vagina, and urethra were associated with age. In bicyclists, there were no correlations between VTs and miles biked per week, duration of riding, or BMI. Composite SPEQ scores indicated normal sexual function in all sexually active subjects. Neither group suffered from sexually related distress. CONCLUSION There is an association between bicycling and decreased genital sensation in competitive women bicyclists. Negative effects on sexual function and quality of life were not apparent in our young, healthy premenopausal cohort.