Vitamin K-dependent carboxylation in ß-cells and diabetes.

Vitamin K is an essential micronutrient and a cofactor for the enzyme γ-glutamyl carboxylase, which adds a carboxyl group to specific glutamic acid residues in proteins transiting through the secretory pathway. Higher vitamin K intake has been linked to a reduced incidence of type 2 diabetes (T2D) in humans. Preclinical work suggests that this effect depends on the γ-carboxylation of specific proteins in ß-cells, including endoplasmic reticulum Gla protein (ERGP), implicated in the control of intracellular Ca2+ levels. In this review we discuss these recent advances linking vitamin K and glucose metabolism, and argue that identification of γ-carboxylated proteins in ß-cells is pivotal to better understand how vitamin K protects from T2D and to design targeted therapies for this disease.

Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in ß cells.

Vitamin K is a micronutrient necessary for γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Recent clinical studies implicate vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse ß cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced ß cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identify endoplasmic reticulum Gla protein (ERGP) as a γ-carboxylated ER-resident Ca2+-binding protein expressed in ß cells. Mechanistically, γ-carboxylation of ERGP protects cells against Ca2+ overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated Ca2+ entry. These results reveal a critical role of vitamin K-dependent carboxylation in regulation of Ca2+ flux in ß cells and in their capacity to adapt to metabolic stress.

Low vitamin K status in adults with cystic fibrosis is associated with reduced body mass index, insulin secretion, and increased pseudomonal colonization.

Patients with cystic fibrosis (CF) are at high risk of fat-soluble vitamin deficiencies, even with supplementation. The contribution of a suboptimal vitamin K status to respiratory and endocrine pathophysiology in CF has been inadequately characterized. This is a cross-sectional study in adult CF patients (≥18 years old) from the Montreal Cystic Fibrosis Cohort. Vitamin K1 (VK1) was measured with high-performance liquid chromatography, using fasted serum samples collected during an oral glucose tolerance test (OGTT: 2 h with plasma glucose and insulin every 30 min) (n = 168). Patients were categorized according to VK1 status (suboptimal defined as <0.30 nmol/L). Suboptimal VK1 levels were observed in 66% of patients. Patients with a suboptimal VK1 status have a higher risk of colonization with Pseudomonas aeruginosa (p = 0.001), have lower body mass index (BMI) (p = 0.003), and were more likely to have exocrine pancreatic insufficiency (p = 0.002). Using an established threshold for VK1, we did show significantly reduced OGTT-derived measures of insulin secretion in patients with a VK1 status below 0.30 nmol/L (first- and second-phase area under the curve (AUC)INS/GLU (p = 0.002 and p = 0.006), AUCINS (p = 0.012) and AUCINS/GLU (p = 0.004)). Subclinical vitamin K deficiency is more common than other fat-soluble vitamin deficiencies in patients with CF. We demonstrate an association between a suboptimal VK1 status and measures of insulin secretion. We highlight the potential associations of mild vitamin K deficiency with pseudomonal colonization and lower BMI, although these need to be validated in prospective studies.

OCA-B does not act as a transcriptional coactivator in T cells.

Pou2af1 encodes for OCA-B, a coactivator of OCT-1/2 transcription factors, which plays a key role in B-cell maturation. The function of OCA-B has also been studied in T cells, where T cells from Pou2af1-/- mice have impaired functions, such as cytokine production and T follicular helper (Tfh) differentiation. Arguably, some of these T-cell phenotypes may result from impaired T-B interactions, secondary to the well-documented B-cell defects in Pou2af1-/- mice. Yet, Pou2af1 is actively transcribed in activated T cells, suggesting a T-cell-intrinsic role. To isolate the T-cell-intrinsic impact of Pou2af1, we generated Pou2af1fl/fl mice with specific genetic disruption of Pou2af1 either in all hematopoietic cells or exclusively in T cells. While we confirm that Pou2af1 is expressed in activated T cells, we surprisingly find that T-cell cytokine production is not impaired in Pou2af1-deficient T cells. Moreover, Pou2af1-sufficient and Pou2af1-deficient T cells have comparable transcriptome profiles, arguing against a T-cell-intrinsic role for Pou2af1. In line with these observations, we demonstrate that Tfh maturation is influenced by T-cell-extrinsic deletion of Pou2af1, as observed both in competitive bone marrow chimeras and in Pou2af1fl/fl mice with specific deletion in B cells. Overall, this study provides strong evidence that Pou2af1 does not act as a transcriptional coactivator in T cells, and conclusively demonstrates that loss of OCA-B in B cells indirectly impacts Tfh differentiation, clarifying the role of OCA-B in the immune system.

ERK3-MK5 signaling regulates myogenic differentiation and muscle regeneration by promoting FoxO3 degradation.

The physiological functions and downstream effectors of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain to be characterized. We recently reported that mice expressing catalytically-inactive ERK3 (Mapk6KD/KD ) exhibit a reduced postnatal growth rate as compared to control mice. Here, we show that genetic inactivation of ERK3 impairs postnatal skeletal muscle growth and adult muscle regeneration after injury. Loss of MAPK-activated protein kinase 5 (MK5) phenocopies the muscle phenotypes of Mapk6KD/KD mice. At the cellular level, genetic or pharmacological inactivation of ERK3 or MK5 induces precocious differentiation of C2C12 or primary myoblasts, concomitant with MyoD activation. Reciprocally, ectopic expression of activated MK5 inhibits myogenic differentiation. Mechanistically, we show that MK5 directly phosphorylates FoxO3, promoting its degradation and reducing its association with MyoD. Depletion of FoxO3 rescues in part the premature differentiation of C2C12 myoblasts observed upon inactivation of ERK3 or MK5. Our findings reveal that ERK3 and its substrate MK5 act in a linear signaling pathway to control postnatal myogenic differentiation.

In Vivo Analysis of the Contribution of Proprotein Convertases to the Processing of FGF23.

Fibroblast growth factor 23 (FGF23) is a hormone secreted from fully differentiated osteoblasts and osteocytes that inhibits phosphate reabsorption by kidney proximal tubules. The full-length (i.e., intact) protein mediates FGF23 endocrine functions, while endoproteolytic cleavage at a consensus cleavage sequence for the proprotein convertases (PCs) inactivates FGF23. Two PCs, furin and PC5, were shown to cleave FGF23 in vitro at RHTR179↓, but whether they are fulfilling this function in vivo is currently unknown. To address this question, we used here mice lacking either or both furin and PC5 in cell-specific manners and mice lacking the paired basic amino acid-cleaving enzyme 4 (PACE4) in all cells. Our analysis shows that furin inactivation in osteoblasts and osteocytes results in a 25% increase in circulating intact FGF23, without any significant impact on serum phosphate levels, whether mice are maintained on a normal or a low phosphate diet. Under conditions of iron deficiency, FGF23 is normally processed in control mice, but its processing is impaired in mice lacking furin in osteoblasts and osteocytes. In contrast, FGF23 is normally cleaved following erythropoietin or IL-1ß injections in mice lacking furin or both furin and PC5, and in PACE4-deficient mice. Altogether, these studies suggest that furin is only partially responsible for FGF23 cleavage under certain conditions in vivo. The processing of FGF23 may therefore involve the redundant action of multiple PCs or of other peptidases in osteoblasts, osteocytes and hematopoietic cells.

Male but not female mice with severe osteogenesis imperfecta are partially protected from high-fat diet-induced obesity.

Previously we have shown that young mice with a dominant severe form of osteogenesis imperfecta (OI), caused by mutated collagen type I, exhibit an altered glucose/insulin metabolism and energy expenditure along with elevated levels of osteocalcin, a bone-derived hormone involved in the regulation of whole-body metabolism. This study aimed to examine the long-term effects of a western diet in these OI mice. Male and female OI mice and wild type littermates (WT) were fed a high-fat diet (HFD) or a matched low-fat diet (LFD) for 26 weeks. HFD-induced obesity was observed in male and female WT and female OI mice, but not in male OI mice. HFD-fed WT and OI mice of both sexes developed hyperglycemia and glucose intolerance, but the degree of glucose intolerance was significantly lower in male and female OI mice compared to sex- and diet-matched WT mice. Indirect calorimetry revealed increased movement of male OI mice on HFD compared to LFD and, while HFD lowered energy expenditure in WT mice, energy expenditure was not changed in OI mice. Further, HFD-fed male OI mice demonstrated a diet-induced increased expression of the thermogenesis genes, Ucp1 and Pgc1α, in brown adipose tissue. On LFD, total and Gla-13 osteocalcin levels were similar in 30-week-old WT and OI mice, but on HFD, both were significantly higher in OI mice than WT. Thus, male OI mice respond to HFD with increased movement, energy expenditure, brown adipose tissue thermogenesis, and higher levels of osteocalcin, resulting in partial protection against HFD-induced obesity.

Gain-of-Function Lrp5 Mutation Improves Bone Mass and Strength and Delays Hyperglycemia in a Mouse Model of Insulin-Deficient Diabetes.

High fracture rate and high circulating levels of the Wnt inhibitor, sclerostin, have been reported in diabetic patients. We studied the effects of Wnt signaling activation on bone health in a mouse model of insulin-deficient diabetes. We introduced the sclerostin-resistant Lrp5A214V mutation, associated with high bone mass, in mice carrying the Ins2Akita mutation (Akita), which results in loss of beta cells, insulin deficiency, and diabetes in males. Akita mice accrue less trabecular bone mass with age relative to wild type (WT). Double heterozygous Lrp5A214V /Akita mutants have high trabecular bone mass and cortical thickness relative to WT animals, as do Lrp5A214V single mutants. Likewise, the Lrp5A214V mutation prevents deterioration of biomechanical properties occurring in Akita mice. Notably, Lrp5A214V /Akita mice develop fasting hyperglycemia and glucose intolerance with a delay relative to Akita mice (7 to 8 vs. 5 to 6 weeks, respectively), despite lack of insulin production in both groups by 6 weeks of age. Although insulin sensitivity is partially preserved in double heterozygous Lrp5A214V /Akita relative to Akita mutants up to 30 weeks of age, insulin-dependent phosphorylated protein kinase B (pAKT) activation in vitro is not altered by the Lrp5A214V mutation. Although white adipose tissue depots are equally reduced in both compound and Akita mice, the Lrp5A214V mutation prevents brown adipose tissue whitening that occurs in Akita mice. Thus, hyperactivation of Lrp5-dependent signaling fully protects bone mass and strength in prolonged hyperglycemia and improves peripheral glucose metabolism in an insulin independent manner. Wnt signaling activation represents an ideal therapeutic approach for diabetic patients at high risk of fracture. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The half-life of the bone-derived hormone osteocalcin is regulated through O-glycosylation in mice, but not in humans.

Osteocalcin (OCN) is an osteoblast-derived hormone with pleiotropic physiological functions. Like many peptide hormones, OCN is subjected to post-translational modifications (PTMs) which control its activity. Here, we uncover O-glycosylation as a novel PTM present on mouse OCN and occurring on a single serine (S8) independently of its carboxylation and endoproteolysis, two other PTMs regulating this hormone. We also show that O-glycosylation increases OCN half-life in plasma ex vivo and in the circulation in vivo. Remarkably, in human OCN (hOCN), the residue corresponding to S8 is a tyrosine (Y12), which is not O-glycosylated. Yet, the Y12S mutation is sufficient to O-glycosylate hOCN and to increase its half-life in plasma compared to wildtype hOCN. These findings reveal an important species difference in OCN regulation, which may explain why serum concentrations of OCN are higher in mouse than in human.

Bones provide support and protection for organs in the body. However, over the last 15 years researchers have discovered that bones also release chemicals known as hormones, which can travel to other parts of the body and cause an effect. The cells responsible for making bone, known as osteoblasts, produce a hormone called osteocalcin which communicates with a number of different organs, including the pancreas and brain. When osteocalcin reaches the pancreas, it promotes the release of another hormone called insulin which helps regulate the levels of sugar in the blood. Osteocalcin also travels to other organs such as muscle, where it helps to degrade fats and sugars that can be converted into energy. It also has beneficial effects on the brain, and has been shown to aid memory and reduce depression. Osteocalcin has largely been studied in mice where levels are five to ten times higher than in humans. But it is unclear why this difference exists or how it alters the role of osteocalcin in humans. To answer this question, Al Rifai et al. used a range of experimental techniques to compare the structure and activity of osteocalcin in mice and humans. The experiments showed that mouse osteocalcin has a group of sugars attached to its protein structure, which prevent the hormone from being degraded by an enzyme in the blood. Human osteocalcin has a slightly different protein sequence and is therefore unable to bind to this sugar group. As a result, the osteocalcin molecules in humans are less stable and cannot last as long in the blood. Al Rifai et al. showed that when human osteocalcin was modified so the sugar group could attach, the hormone was able to stick around for much longer and reach higher levels when added to blood in the laboratory. These findings show how osteocalcin differs between human and mice. Understanding this difference is important as the effects of osteocalcin mean this hormone can be used to treat diabetes and brain disorders. Furthermore, the results reveal how the stability of osteocalcin could be improved in humans, which could potentially enhance its therapeutic effect.

Targeting Bone Cells During Sexual Maturation Reveals Sexually Dimorphic Regulation of Endochondral Ossification.

In endochondral ossification, chondroblasts become embedded in their matrix and become chondrocytes, which are mature cells that continue to proliferate, eventually becoming hypertrophic. Hypertrophic chondrocytes produce cartilage that is then resorbed by osteoclasts prior to bone matrix replacement via osteoblasts. Although sexually dimorphic bone phenotypes have long been characterized, specific modulation of the growth plate during a critical window in sexual maturation has not been evaluated. Here we report that specific depletion of osteocalcin- (OCN-) expressing cells in vivo during sexual maturation leads to dimorphic bone phenotypes in males and females. At 6 to 8 weeks of age, OCN-Cre;iDTR (inducible diphtheria toxin receptor-expressing) mice were treated with diphtheria toxin (DT) for 2 weeks to deplete OCN+ cells. At the end of the study, long bones were collected for µCT and histomorphometry, and serum was collected for proteomic and lipidomic analyses. Ablation of OCN+ cells in mice leads to consistent trends for weight loss after 2 weeks of treatment. Females exhibited decreased skeletal parameters in response to OCN+ cell ablation treatment, as expected. However, OCN+ cell ablation in males uniquely displayed an expansion of hypertrophic chondrocytes, a widening of the growth plate, and an abnormal "clubbing" anatomy of the distal femur. Following DT treatment, mice from both sexes also underwent metabolic cage analysis, in which both sexes exhibited decreased energy expenditure. We conclude that skewing endochondral bone formation during longitudinal growth has a profound effect on body weight and energy expenditure with sex-specific effects on developing bone. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

PHOSPHO1 is a skeletal regulator of insulin resistance and obesity.

BACKGROUND: The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have identified the skeleton as a key endocrine regulator of metabolism. The bone-specific phosphatase, Phosphatase, Orphan 1 (PHOSPHO1), which is indispensable for bone mineralisation, has been recently implicated in the regulation of energy metabolism in humans, but its role in systemic metabolism remains unclear. Here, we probe the mechanism underlying metabolic regulation by analysing Phospho1 mutant mice. RESULTS: Phospho1-/- mice exhibited improved basal glucose homeostasis and resisted high-fat-diet-induced weight gain and diabetes. The metabolic protection in Phospho1-/- mice was manifested in the absence of altered levels of osteocalcin. Osteoblasts isolated from Phospho1-/- mice were enriched for genes associated with energy metabolism and diabetes; Phospho1 both directly and indirectly interacted with genes associated with glucose transport and insulin receptor signalling. Canonical thermogenesis via brown adipose tissue did not underlie the metabolic protection observed in adult Phospho1-/- mice. However, the decreased serum choline levels in Phospho1-/- mice were normalised by feeding a 2% choline rich diet resulting in a normalisation in insulin sensitivity and fat mass. CONCLUSION: We show that mice lacking the bone mineralisation enzyme PHOSPHO1 exhibit improved basal glucose homeostasis and resist high-fat-diet-induced weight gain and diabetes. This study identifies PHOSPHO1 as a potential bone-derived therapeutic target for the treatment of obesity and diabetes.

AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion protein network.

Aberrant expression of receptor tyrosine kinase AXL is linked to metastasis. AXL can be activated by its ligand GAS6 or by other kinases, but the signaling pathways conferring its metastatic activity are unknown. Here, we define the AXL-regulated phosphoproteome in breast cancer cells. We reveal that AXL stimulates the phosphorylation of a network of focal adhesion (FA) proteins, culminating in faster FA disassembly. Mechanistically, AXL phosphorylates NEDD9, leading to its binding to CRKII which in turn associates with and orchestrates the phosphorylation of the pseudo-kinase PEAK1. We find that PEAK1 is in complex with the tyrosine kinase CSK to mediate the phosphorylation of PAXILLIN. Uncoupling of PEAK1 from AXL signaling decreases metastasis in vivo, but not tumor growth. Our results uncover a contribution of AXL signaling to FA dynamics, reveal a long sought-after mechanism underlying AXL metastatic activity, and identify PEAK1 as a therapeutic target in AXL positive tumors.

Association between changes in bioactive osteocalcin and glucose homeostasis after biliopancreatic diversion.

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Measurement of bioactive osteocalcin in humans using a novel immunoassay reveals association with glucose metabolism and ß-cell function.

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 ß-cell dysfunction in humans.

VKORC1L1, An Enzyme Mediating the Effect of Vitamin K in Liver and Extrahepatic Tissues.

Vitamin K is an essential nutrient involved in the regulation of blood clotting and tissue mineralization. Vitamin K oxidoreductase (VKORC1) converts vitamin K epoxide into reduced vitamin K, which acts as the co-factor for the γ-carboxylation of several proteins, including coagulation factors produced by the liver. VKORC1 is also the pharmacological target of warfarin, a widely used anticoagulant. Vertebrates possess a VKORC1 paralog, VKORC1-like 1 (VKORC1L1), but until very recently, the importance of VKORC1L1 for protein γ-carboxylation and hemostasis in vivo was not clear. Here, we first review the current knowledge on the structure, function and expression pattern of VKORC1L1, including recent data establishing that, in the absence of VKORC1, VKORC1L1 can support vitamin K-dependent carboxylation in the liver during the pre- and perinatal periods in vivo. We then provide original data showing that the partial redundancy between VKORC1 and VKORC1L1 also exists in bone around birth. Recent studies indicate that, in vitro and in cell culture models, VKORC1L1 is less sensitive to warfarin than VKORC1. Genetic evidence is presented here, which supports the notion that VKORC1L1 is not the warfarin-resistant vitamin K quinone reductase present in the liver. In summary, although the exact physiological function of VKORC1L1 remains elusive, the latest findings clearly established that this enzyme is a vitamin K oxidoreductase, which can support γ-carboxylation in vivo.

Loss of OcaB Prevents Age-Induced Fat Accretion and Insulin Resistance by Altering B-Lymphocyte Transition and Promoting Energy Expenditure.

The current demographic shift toward an aging population has led to a robust increase in the prevalence of age-associated metabolic disorders. Recent studies have demonstrated that the etiology of obesity-related insulin resistance that develops with aging differs from that induced by high-calorie diets. Whereas the role of adaptive immunity in changes in energy metabolism driven by nutritional challenges has recently gained attention, its impact on aging remains mostly unknown. Here we found that the number of follicular B2 lymphocytes and expression 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 IgG and impaired glucose homeostasis. Reduction of B-cell maturation and Ig 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 the 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.

VKOR paralog VKORC1L1 supports vitamin K-dependent protein carboxylation in vivo.

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.

Regulation of Energy Metabolism by Bone-Derived Hormones.

Like many other organs, bone can act as an endocrine organ through the secretion of bone-specific hormones or "osteokines." At least two osteokines are implicated in the control of glucose and energy metabolism: osteocalcin (OCN) and lipocalin-2 (LCN2). OCN stimulates the production and secretion of insulin by the pancreatic ß-cells, but also favors adaptation to exercise by stimulating glucose and fatty acid (FA) utilization by the muscle. Both of these OCN functions are mediated by the G-protein-coupled receptor GPRC6A. In contrast, LCN2 influences energy metabolism by activating appetite-suppressing signaling in the brain. This action of LCN2 occurs through its binding to the melanocortin 4 receptor (MC4R) in the paraventricular nucleus of the hypothalamus (PVN) and ventromedial neurons of the hypothalamus.