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Bone may regulate glucose homeostasis via uncarboxylated bioactive osteocalcin (ucOCN). This study explored whether changes in ucOCN and bone remodeling are associated with change in glucose homeostasis after biliopancreatic diversion (BPD). In this secondary exploratory analysis of a 1-year prospective observational study, 16 participants (11 men/5 women; 69% with type 2 diabetes; mean BMI 49.4 kg/m2) were assessed before, 3 days, 3 months and 12 months after BPD. Changes in plasma ucOCN and bone markers (C-terminal telopeptide (CTX), total osteocalcin (OCN)) were correlated with changes in insulin resistance or sensitivity indices (HOMA-IR; adipose tissue insulin resistance index (ADIPO-IR) and insulin sensitivity index (SI) from the hyperinsulinemic-euglycemic clamp), insulin secretion rate (ISR) from the hyperglycemic clamp, and disposition index (DI: SI × ISR) using Spearman correlations before and after adjustment for weight loss. ucOCN was unchanged at 3 days but increased dramatically at 3 months (+257%) and 12 months (+498%). Change in ucOCN correlated significantly with change in CTX at 3 months (r = 0.62, p = 0.015) and 12 months (r = 0.64, p = 0.025) before adjustment for weight loss. It also correlated significantly with change in fasting insulin (r = −0.53, p = 0.035), HOMA-IR (r = −0.54, p = 0.033) and SI (r = 0.52, p = 0.041) at 3 days, and ADIPO-IR (r = −0.69, p = 0.003) and HbA1c (r = −0.69, p = 0.005) at 3 months. Change in OCN did not correlate with any glucose homeostasis indices. Results were similar after adjustment for weight loss. The increase in ucOCN may be associated with the improvement in insulin resistance after BPD, independently of weight loss. These findings need to be confirmed in larger, less heterogeneous populations.

The current demographic shift toward an aging population has led to a robust increase in the prevalence of age-associated metabolic disorders. Interestingly, recent studies have demonstrated that the etiology of obesity-related insulin resistance developed upon aging differs from that induced by high-calorie diets. Whereas the role of adaptive immunity on the changes in energy metabolism driven by nutritional challenges has gained attention in the past years, its impact upon aging remains mostly unknown. Here, we found that follicular B2 lymphocytes and expression levels of the B cell-specific transcriptional coactivator OcaB increase with age in spleen and in intra-abdominal epididymal white adipose tissue (eWAT), concomitantly with higher circulating levels of immunoglobulin G (IgG) and impaired glucose homeostasis. Reduction of B cell maturation and immunoglobulin production - especially that of IgG2c - by ablation of OcaB prevented age-induced glucose intolerance and insulin resistance, and promoted energy expenditure by stimulating fatty acid utilization in eWAT and brown adipose tissue. Transfer of wild-type bone marrow in OcaB-/- mice replenished eWAT B2 cell population and IgG levels, which diminished glucose tolerance, insulin sensitivity, and energy expenditure, while increasing body weight gain in aged mice. Thus, these findings demonstrate that upon aging, modifications in B cell-driven adaptive immunity contribute to glucose intolerance and fat accretion.

Osteocalcin (OCN) is a bone-derived hormone involved in the regulation of glucose metabolism. In serum, OCN exists in carboxylated and uncarboxylated forms (ucOCN), and studies in rodents suggest that ucOCN is the bioactive form of this hormone. Whether this is also the case in humans is unclear, because a reliable assay to measure ucOCN is not available. Here, we established and validated a new immunoassay (ELISA) measuring human ucOCN and used it to determine the level of bioactive OCN in two cohorts of overweight or obese subjects, with or without type 2 diabetes (T2D). The ELISA could specifically detect ucOCN concentrations ranging from 0.037 to 1.8 ng/mL. In a first cohort of overweight or obese postmenopausal women without diabetes ( n = 132), ucOCN correlated negatively with fasting glucose (r = −0.18, P = 0.042) and insulin resistance assessed by the homeostatic model assessment of insulin resistance (r = −0.18, P = 0.038) and positively with insulin sensitivity assessed by a hyperinsulinemic-euglycemic clamp (r = 0.18, P = 0.043) or insulin sensitivity index derived from an oral glucose tolerance test (r = 0.26, P = 0.003). In a second cohort of subjects with severe obesity ( n = 16), ucOCN was found to be lower in subjects with T2D compared with those without T2D (2.76 ± 0.38 versus 4.52 ± 0.06 ng/mL, P = 0.009) and to negatively correlate with fasting glucose (r = −0.50, P = 0.046) and glycated hemoglobin (r = −0.57, P = 0.021). Moreover, the subjects with ucOCN levels below 3 ng/mL had a reduced insulin secretion rate during a hyperglycemic clamp ( P = 0.03). In conclusion, ucOCN measured with this novel and specific assay is inversely associated with insulin resistance and β-cell dysfunction in humans.

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., Osteocalcin (OCN) is an osteoblast-derived hormone favoring glucose homeostasis, energy expenditure, male fertility, brain development, and cognition. Before being secreted by osteoblasts in the bone extracellular matrix, OCN is γ-carboxylated by the γ-carboxylase (GGCX) on three glutamic acid residues, a cellular process requiring reduction of vitamin K (VK) by a second enzyme, a reductase called VKORC1. Although circumstantial evidence suggests that γ-carboxylation may inhibit OCN endocrine functions, genetic evidence that it is the case is still lacking. Here we show using cell-specific gene inactivation models that γ-carboxylation of OCN by GGCX inhibits its endocrine function. We further show that VKORC1 is required for OCN γ-carboxylation in osteoblasts, whereas its paralogue, VKORC1L1, is dispensable for this function and cannot compensate for the absence of VKORC1 in osteoblasts. This study genetically and biochemically delineates the functions of the enzymes required for OCN modification and demonstrates that it is the uncarboxylated form of OCN that acts as a hormone.

Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K-dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1-/- mice survived longer (1 week) than Ggcx-/- mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1-/-;Vkorc1l1-/- mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1-/- mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1-/- mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.

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.

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.

The only well described function of vitamin K (VK) is to serve as a co-factor for the γ-carboxylation of particular proteins to convert specific glutamic acid (Glu) residues to γ-carboxyglutamic acid (Gla) residues. This process involves two enzymes, γ-glutamyl carboxylase (γ-carboxylase or GGCX) and vitamin K epoxide reductase (VKORC1), which together constitute the vitamin K cycle [1]. Physiologically, γ-carboxylation is known to be critical for the function of many coagulation factors (prothrombin, factor IX, factor VII, etc), but is also implicated in the regulation of Matrix Gla Protein (MGP) that inhibits extra-osseous tissue mineralization and of osteocalcin that regulates glucose metabolism. Osteocalcin is an osteoblast-derived γ-carboxylated protein present at high concentration in the bone extracellular matrix (ECM). Because Gla osteocalcin have great affinity for hydroxyapatite, the mineral component of bone ECM, it was originally believed to play a role in bone mineralization. Nevertheless, gain- and loss-of-function mouse models of osteocalcin have shown that this protein is not required for normal bone remodeling or mineralization [2]. Instead, the characterization of Ocn−/− mice revealed that osteocalcin is acting as a bone-derived hormone regulating glucose metabolism [3-4]. Cell culture and in vivo studies have shown that osteocalcin improves glucose handling by promoting insulin secretion by β-cells on one hand and by favouring insulin sensitivity on the other hand [1]. Although both the Gla and the Glu forms of osteocalcin are detected in the serum, the vast majority of the in vitro and in vivo studies conducted so far suggests that the endocrine function of osteocalcin is fulfilled by its Glu form. Interestingly, additional experiments involving the characterization of mice having either increased or decreased osteoclast number suggest that bone resorption is required to decarboxylate and release osteocalcin from the bone matrix [5–6]. Accordingly, many but not all studies in human have shown that serum levels of Glu osteocalcin negatively correlate with insulin resistance, obesity, diabetes or markers of the metabolic syndrome [1]. So far the data on osteocalcin endocrine regulation were supporting a model in which osteocalcin produced by osteoblasts is stored as a γ-carboxylated and inactive protein in the bone ECM, before being activated by decarboxylation during bone resorption. This model predicts that blocking osteocalcin γ-carboxylation would prevent osteocalcin accumulation in the bone ECM and thereby improve glucose homeostasis. In a recent study published in The Journal of Cell Biology [7] we tested directly this assumption aiming at providing direct evidence demonstrating that γ-carboxylation acts negatively on the endocrine functions of osteocalcin. Mice lacking Ggcx or Vkorc1 globally die perinatally from severe hemorrhages, precluding the study of the role of γ-carboxylation and VK reduction on osteocalcin endocrine function in adults. Hence we generated “floxed” alleles of Ggcx and Vkorc1 in mice in order to be able to inactivate each of these two genes in a tissue-specific manner. Interestingly, mice lacking Ggcx or Vkorc1 in osteoblasts were viable and displayed a marked reduction in Gla osteocalcin serum levels while Glu osteocalcin levels were increased. In addition, osteocalcin failed to accumulate in the bone ECM in the Ggcx and Vkorc1 osteoblast-specific knockout mice. Importantly, these mice also show a significant improvement in glucose tolerance and in insulin sensitivity and were protected from high fat diet-induced insulin resistance and obesity [7]. There is only one γ-carboxylase-encoding gene in mammals; however, all vertebrates possess a second VKORC1-like encoding gene called VKORC1L1. The function of this enzyme remains unclear, but it has been suggested to have VK epoxide reductase activity in vitro, compensating for the absence of VKORC1 in particular cell culture conditions. For this reason, we also generated a conditional allele of this gene in the mouse. Our characterization of the global and osteoblast-specific Vkorc1l1 deficient mice suggests that VKORC1L1 is not implicated in osteocalcin carboxylation. In addition, deleting Vkorc1l1 in osteoblast-specific Vkorc1-deficient mice did not further affect the circulating levels of Glu osteocalcin or osteocalcin bone content when compared to Vkorc1-osteoblasts knockout littermates [7]. These results suggest that VKORC1L1 cannot compensate for VKORC1 absence with respect to γ-carboxylation in osteoblasts. However, it cannot be excluded that VKORC1 and VKORC1L1 may have redundant function(s) in osteoblasts independently of γ-carboxylation. Altogether, these studies support the conclusion that, at least in mice, Glu osteocalcin is the active form of this hormone in vivo and that γ-carboxylation negatively regulates both the bioactivity and the bioavailability of osteocalcin. In addition, this work demonstrates a novel role for vitamin K-dependent γ-carboxylation in the control of glucose metabolism and suggests that VKORC1L1 does not act as a VK reductase in vivo. Future studies will be oriented toward understanding the physiological function of VKORC1L1 in bone and in other tissues.

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 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. Guess MK, Connell K, Schrader S, Reutman S, Wang A, LaCombe J, Toennis C, Lowe B, Melman A, and Mikhail MK. Genital sensation and sexual function in women bicyclists and runners: Are your feet safer than your seat? J Sex Med 2006;3:1018–1027.

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.

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 osteocalcin's 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.

Introduction Cycling is associated with genital neuropathies and erectile dysfunction in males. Women riders also have decreased genital sensation; however, sparse information exists addressing the effects of modifiable risks on neurological injuries in females. Aim This study assesses the effects of bicycle setup and cyclists' attributes on GS and saddle pressures among female cyclists. Methods Previously, we compared genital sensation in competitive female cyclists (N = 48) to that of female runners (N = 22). The current study is a subanalysis of the 48 cyclists from the original study group. Nonpregnant, premenopausal women who rode at least 10 miles per week, 4 weeks per month were eligible for participation. Main Outcome Measures Genital sensation was measured in microns using biosthesiometry measures of vibratory thresholds (VTs). Perineal and total saddle pressures were determined using a specialized pressure map and recorded in kilopascals (kPA). Results Handlebars positioned lower than the saddle correlated with increased perineum saddle pressures and decreased anterior vaginal and left labial genital sensation ( P P P P P Conclusion Handlebars positioned lower than the saddle were significantly associated with increased perineum saddle pressures and decreased genital sensation in female cyclists. Modifying bicycle setup may help alleviate neuropathies in females. Additional research is warranted to further assess the extent of the associations. Partin SN, Connell KA, Schrader S, LaCombe J, Lowe B, Sweeney A, Reutman S, Wang A, Toennis C, Melman A, Mikhail M, and Guess MK. The bar sinister: Does handlebar level damage the pelvic floor in female cyclists? J Sex Med 2012;9:1367–1373.

Introduction There are numerous genital complaints in women cyclists, including pain, numbness, and edema of pelvic floor structures. Debate ensues about the best saddle design for protection of the pelvic floor. Aim To investigate the relationships between saddle design, seat pressures, and genital nerve function in female, competitive cyclists. Methods We previously compared genital sensation in healthy, premenopausal, competitive women bicyclists and runners. The 48 cyclists from our original study comprise the study group in this subanalysis. Main Outcome Measures Main outcome measures were: (i) genital vibratory thresholds (VTs) determined using the Medoc Vibratory Sensation Analyzer 3000 and (ii) saddle pressures as determined using a specially designed map sensor. Results More than half of the participants (54.8%) used traditional saddles, and the remainder (45.2%) rode with cut-out saddles. On bivariate analysis, use of traditional saddles was associated with lower mean perineal saddle pressures (MPSP) than riding on cut-out saddles. Peak perineal saddle pressures (PPSP) were also lower; however, the difference did not reach statistical significance. Saddle design did not affect mean or peak total saddle pressures (MTSP, PTSP). Saddle width was significantly associated with PPSP, MTSP, and PTSP but not with MPSP. Women riding cut-out saddles had, on average, a 4 and 11 kPa increase in MPSP and PPSP, respectively, compared with women using traditional saddles (P = 0.008 and P = 0.010), after adjustment for other variables. Use of wider saddles was associated with lower PPSP and MTSP after adjustment. Although an inverse correlation was seen between saddle pressures and VTs on bivariate analysis, these differences were not significant after adjusting for age. Conclusion Cut-out and narrower saddles negatively affect saddle pressures in female cyclists. Effects of saddle design on pudendal nerve sensory function were not apparent in this cross-sectional analysis. Longitudinal studies evaluating the long-term effects of saddle pressure on the integrity of the pudendal nerve, pelvic floor, and sexual function are warranted.