Circulating lipocalin-2 and features of metabolic syndrome in community-dwelling older women: A cross-sectional study.

Lipocalin-2 (LCN2) is released by several cell types including osteoblasts and adipocytes and has been suggested as a marker of renal dysfunction, metabolic syndrome (MetS) and type 2 diabetes (T2D). Whether LCN2 is linked to these diseases in older women remains unknown. This study investigated whether LCN2 is related to features of MetS and T2D in older women. This cross-sectional study included 705 non-diabetic women (mean age 75.1 ± 2.6 years) for MetS analysis and 76 women (mean age 75.4 ± 2.8 years) with T2D. Total circulating LCN2 levels were analysed using a two-step chemiluminescent microparticle monoclonal immunoassay. MetS was determined by a modified National Cholesterol Education Program Adult Treatment Panel III classification. Multivariable-adjusted logistic regression analysis was used to assess odds ratios between LCN2 quartiles and MetS. Women in the highest LCN2 quartile had approximately 3 times greater risk for MetS compared to women in the lowest quartile (OR 3.05; 95%CI 1.86-5.02). Women with T2D or MetS scores of ≥ 3 had higher LCN2 levels compared to women with a MetS score of 0 (p < 0.05). Higher LCN2 correlated with higher body mass index, fat mass, triglycerides and glycated haemoglobin and lower high-density lipoprotein cholesterol and estimated glomerular filtration rate (p < 0.05). Higher circulating levels of LCN2 are associated with worsened cardio-metabolic risk factors and increased odds of MetS and T2D in older women. Whether it can be used as a biomarker for identifying those at risk for MetS and T2D should be explored further.

Undercarboxylated osteocalcin and ibandronate combination ameliorates hindlimb immobilization-induced muscle wasting.

Immobilization leads to muscle wasting and insulin resistance, particularly during ageing. It has been suggested that undercarboxylated osteocalcin (ucOC) improves muscle mass and glucose metabolism. Bisphosphonates, an anti-osteoporosis treatment, might protect muscle wasting independent of ucOC. We hypothesize that the combination of ucOC and ibandronate (IBN) treatments has superior protective effects against immobilization-induced muscle wasting and insulin resistance than either treatment alone. C57BL/6J mice were hindlimb-immobilized for two weeks, with injections of vehicle, ucOC (90 ng/g daily) and/or IBN (2 µg/g weekly). Insulin/oral glucose tolerance tests (ITT/OGTT) were performed. Immediately after immobilization, muscles (extensor digitorum longus (EDL), soleus, tibialis anterior, gastrocnemius and quadriceps) were isolated and measured for muscle mass. Insulin-stimulated glucose uptake (EDL and soleus) was examined. Phosphorylation/expression of proteins in anabolic/catabolic pathways were examined in quadriceps. Primary human myotubes derived from older adult muscle biopsies were treated with ucOC and/or IBN, then signalling proteins were analysed. Combined treatment, but not individual treatments, significantly increased the muscle weight/body weight ratio in immobilized soleus (31.7%; P = 0.013) and quadriceps (20.0%; P = 0.0008) muscles, concomitant with elevated p-Akt (S473)/Akt ratio (P = 0.0047). Combined treatment also enhanced whole-body glucose tolerance (16.6%; P = 0.0011). In human myotubes, combined treatment stimulated greater activation of ERK1/2 (P = 0.0067 and 0.0072) and mTOR (P = 0.036), and led to a lesser expression of Fbx32 (P = 0.049) and MuRF1 (P = 0.048) than individual treatments. These findings suggest a potential therapeutic role for the ucOC and bisphosphonates combination in protecting against muscle wasting induced by immobilization and ageing. KEY POINTS: It has been suggested that undercarboxylated osteocalcin (ucOC) improves muscle mass and glucose metabolism. Bisphosphonates, an anti-osteoporosis treatment, might protect against muscle wasting independent of ucOC. The combination treatment of ucOC and ibandronate was shown to exert a greater therapeutic effect against immobilization-induced muscle wasting, and led to greater activation of anabolic pathway and less expression of catabolic signalling proteins in myotubes derived from older adults, compared with individual treatments. The combination treatment was found to improve whole-body glucose tolerance. Our findings suggest a potential therapeutic role for the ucOC and bisphosphonates combination in protecting against muscle wasting induced by immobilization and ageing.

Bone Turnover Markers Including Undercarboxylated Osteocalcin Are Associated With Mortality Risk in Older Men.

Osteocalcin in its undercarboxylated form (ucOC) may influence diabetes risk; however, its relationship with all-cause and cause-specific mortality is unclear. Whether other bone turnover markers (BTMs) are associated with mortality risk differently from ucOC also remains uncertain. Our aim was to determine associations of serum ucOC with all-cause and cause-specific mortality and compare these with the corresponding associations of serum total osteocalcin (TOC), procollagen type I N-propeptide (PINP), and collagen type 1 C-terminal cross-linked telopeptide (CTX) in older men. We conducted a prospective cohort study of 3871 community-dwelling men, aged 77.0 ± 3.6 years at baseline, followed for a median of 12.3 years. Exposure variables were ucOC, TOC, PINP, and CTX concentrations assayed in serum. Outcomes were incidence of all deaths and deaths due to cardiovascular disease (CVD) or cancer, ascertained using death registry data. Cox regression analyses adjusted for cardiovascular risk factors and prevalent CVD and for prevalent cancer in analyses of cancer-related mortality. Higher concentrations of ucOC, PINP, and CTX were associated with all-cause mortality (hazard ratio [HR] per 1 standard deviation increase: ucOC 1.12, 95% confidence interval [CI] 1.06-1.18, p < 0.001; PINP HR = 1.06, 95% CI 1.01-1.11, p = 0.009; CTX HR = 1.13, 95% CI 1.08-1.19, p < 0.001), but TOC was not associated. Similar results were found after excluding men with an incident fracture during follow-up. Higher ucOC and CTX were associated with CVD mortality (ucOC HR per 1 SD increase 1.13, 95% CI 1.05-1.22, p = 0.001; CTX HR = 1.12, 95% CI 1.04-1.20, p = 0.003), but this result was not significant in competing risks analysis. Higher CTX was also associated with cancer mortality (HR = 1.12, 95% CI 1.01-1.23, p = 0.024). In conclusion, in older men, higher bone turnover, assessed by BTMs including ucOC, is a biomarker for all-cause mortality risk. Undercarboxylated osteocalcin was a more informative biomarker for this outcome than TOC. Higher CTX was associated with all-cause and cancer-related mortality. Further evaluation of causality and potential underlying mechanisms is warranted. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Prior aerobic exercise mitigates the decrease in serum osteoglycin and lipocalin-2 following high-glucose mixed-nutrient meal ingestion in young men.

Osteoglycin (OGN) and lipocalin-2 (LCN2) are hormones that can be secreted by bone and have been linked to glucose homeostasis in rodents. However, the endocrine role of these hormones in humans is contradictory and unclear. We examined the effects of exercise and meal ingestion on circulating serum OGN and LCN2 levels in eight healthy males {age: 28 [25, 30] years [median ± interquartile range (IQR)] and body mass index [BMI]: 24.3 [23.6, 25.5] kg/m2}. In a randomized crossover design, participants ingested a high-glucose (1.1 g glucose/kg body wt) mixed-nutrient meal (45% carbohydrate, 20% protein, and 35% fat) on a rest-control day and 3 and 24 h after aerobic cycling exercise (1 h at 70%-75% V̇o2peak). Acute aerobic exercise increased serum LCN2 levels immediately after exercise (∼61%), which remained elevated 3-h postexercise (∼55%). In contrast, serum OGN remained similar to baseline levels throughout the 3-h postexercise recovery period. The ingestion of a high-glucose mixed-nutrient meal led to a decrease in serum OGN at 90-min (approximately -17%) and 120-min postprandial (approximately -44%), and a decrease in LCN2 at 120-min postprandial (approximately -26%). Compared with the control meal, prior exercise elevated serum OGN and LCN2 levels at 120-min postprandial when the meal was ingested 3-h (OGN: ∼74% and LCN2: ∼68%) and 24-h postexercise (OGN: ∼56% and LCN2: ∼16%). Acute exercise increases serum LCN2 and attenuates the postprandial decrease in OGN and LCN2 following high-glucose mixed-nutrient meal ingestion. The potential endocrine role of circulating OGN and LCN2 in humans warrants further investigation.NEW & NOTEWORTHY We provide novel evidence that OGN and LCN2 decrease 120 min after ingesting a high-glucose mixed-nutrient meal in healthy adults. Acute aerobic exercise increases circulating LCN2 for up to 3-h postexercise, whereas circulating OGN remains similar to baseline. Despite differing postexercise responses, postprandial LCN2 and OGN are elevated when the high-glucose meal is ingested 3-h and 24-h postexercise. Findings support that OGN and LCN2 are dynamically linked to energy homeostasis in humans.

High-intensity training elicits greater improvements in cardio-metabolic and reproductive outcomes than moderate-intensity training in women with polycystic ovary syndrome: a randomized clinical trial.

STUDY QUESTION: Does 12 weeks of high-intensity interval training (HIIT) result in greater improvements in cardio-metabolic and reproductive outcomes compared to standard moderate-intensity continuous training (MICT) in women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: HIIT offers greater improvements in aerobic capacity, insulin sensitivity and menstrual cyclicity, and larger reductions in hyperandrogenism compared to MICT. WHAT IS KNOWN ALREADY: Exercise training is recognized to improve clinical outcomes in women with PCOS, but little is known about whether HIIT results in greater health outcomes compared to standard MICT. STUDY DESIGN, SIZE, DURATION: This was a two-armed randomized clinical trial enrolling a total of 29 overweight women with PCOS between May 2016 and November 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with PCOS aged 18-45 years were randomly assigned to 12 weeks of either MICT (60-75% peak heart rate, N = 14) or HIIT (>90% peak heart rate, N = 15), each completed three times per week. The primary clinical outcomes were aerobic capacity (VO2peak) and insulin sensitivity (euglycaemic-hyperinsulinaemic clamp). Secondary outcomes included hormonal profiles, menstrual cyclicity and body composition. MAIN RESULTS AND THE ROLE OF CHANCE: Both HIIT and MICT improved VO2peak (HIIT; Δ 5.8 ± 2.6 ml/kg/min, P < 0.001 and MICT; Δ 3.2 ± 2 ml/kg/min, P < 0.001), however, the HIIT group had a greater improvement in aerobic capacity compared to MICT (ß = 2.73 ml/kg/min, P = 0.015). HIIT increased the insulin sensitivity index compared to baseline (Δ 2.3 ± 4.4 AU, P = 0.007) and MICT (ß = 0.36 AU, P = 0.030), and caused higher increases in sex hormone-binding globulin compared to MICT (ß = 0.25 nmol/l, P = 0.002). HIIT participants were 7.8 times more likely to report improved menstrual cyclicity than those in the MICT group (odds ratio 7.8, P = 0.04). LIMITATIONS, REASONS FOR CAUTION: This study has a small sample size and the findings of the effect of the exercise interventions are limited to overweight reproductive-aged women, who do not have any co-existing co-morbidities that require medication. WIDER IMPLICATIONS OF THE FINDINGS: Exercise, regardless of intensity, has clear health benefits for women with PCOS. HIIT appears to be a more beneficial strategy and should be considered for promoting health and reducing cardio-metabolic risk in overweight women with PCOS. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a Project Support Grant from the Australian National Health and Medical Research Council (NHMRC) Centre for Research Excellence in PCOS. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: ACTRN12615000242527. TRIAL REGISTRATION DATE: 19 February 2015. DATE OF FIRST PATIENT'S ENROLMENT: 27 May 2016.

The mitochondrial profile in women with polycystic ovary syndrome: impact of exercise.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting pre-menopausal women and involves metabolic dysregulation. Despite the high prevalence of insulin resistance, the existence of mitochondrial dysregulation and its role in the pathogenesis of PCOS is not clear. Exercise is recommended as the first-line therapy for women with PCOS. In particular, high-intensity interval training (HIIT) is known to improve metabolic health and enhance mitochondrial characteristics. In this narrative review, the existing knowledge of mitochondrial characteristics in skeletal muscle and adipose tissue of women with PCOS and the effect of exercise interventions in ameliorating metabolic and mitochondrial health in these women are discussed. Even though the evidence on mitochondrial dysfunction in PCOS is limited, some studies point to aberrant mitochondrial functions mostly in skeletal muscle, while there is very little research in adipose tissue. Although most exercise intervention studies in PCOS report improvements in metabolic health, they show diverse and inconclusive findings in relation to mitochondrial characteristics. A limitation of the current study is the lack of comprehensive mitochondrial analyses and the diversity in exercise modalities, with only one study investigating the impact of HIIT alone. Therefore, further comprehensive large-scale exercise intervention studies are required to understand the association between metabolic dysfunction and aberrant mitochondrial profile, and the molecular mechanisms underlying the exercise-induced metabolic adaptations in women with PCOS.

Lipocalin-2 Variants and Their Relationship With Cardio-Renal Risk Factors.

Objectives: To investigate the serum, plasma and urine levels of lipocalin-2 (LCN2) variants in healthy humans and their associations with risk factors for cardiometabolic (CMD) and chronic kidney (CKD) diseases. Methods: Fifty-nine males and 41 females participated in the study. Blood and urine were collected following an overnight fasting. LCN2 variants were analyzed using validated in-house ELISA kits. Heart rate, blood pressure, lipids profile, glucose, adiponectin, high-sensitivity C-reactive protein (hsCRP), creatinine, cystatin C, and biomarkers for kidney function were assessed. Results: The levels of hLcn2, C87A and R81E in serum and urine, but not plasma, were significantly higher in men than women. Increased levels of LCN2 variants, as well as their relative ratios, in serum and plasma were positively associated with body mass index, blood pressure, triglyceride and hsCRP (P<0.05). No significant correlations were found between these measures and hLcn2, C87A or R81E in urine. However, LCN2 variants in urine, but not plasma or serum, were correlated with biomarkers of kidney function (P<0.05). Conclusions: Both the serum and plasma levels of LCN2 variants, as well as their ratios are associated with increased cardiometabolic risk, whereas those in urine are correlated with renal dysfunction. LCN2 variants represent promising biomarkers for CMD and CKD.

Distinct Effects of a High Fat Diet on Bone in Skeletally Mature and Developing Male C57BL/6J Mice.

Increased risks of skeletal fractures are common in patients with impaired glucose handling and type 2 diabetes mellitus (T2DM). The pathogenesis of skeletal fragility in these patients remains ill-defined as patients present with normal to high bone mineral density. With increasing cases of glucose intolerance and T2DM it is imperative that we develop an accurate rodent model for further investigation. We hypothesized that a high fat diet (60%) administered to developing male C57BL/6J mice that had not reached skeletal maturity would over represent bone microarchitectural implications, and that skeletally mature mice would better represent adult-onset glucose intolerance and the pre-diabetes phenotype. Two groups of developing (8 week) and mature (12 week) male C57BL/6J mice were placed onto either a normal chow (NC) or high fat diet (HFD) for 10 weeks. Oral glucose tolerance tests were performed throughout the study period. Long bones were excised and analysed for ex vivo biomechanical testing, micro-computed tomography, 2D histomorphometry and gene/protein expression analyses. The HFD increased fasting blood glucose and significantly reduced glucose tolerance in both age groups by week 7 of the diets. The HFD reduced biomechanical strength, both cortical and trabecular indices in the developing mice, but only affected cortical outcomes in the mature mice. Similar results were reflected in the 2D histomorphometry. Tibial gene expression revealed decreased bone formation in the HFD mice of both age groups, i.e., decreased osteocalcin expression and increased sclerostin RNA expression. In the mature mice only, while the HFD led to a non-significant reduction in runt-related transcription factor 2 (Runx2) RNA expression, this decrease became significant at the protein level in the femora. Our mature HFD mouse model more accurately represents late-onset impaired glucose tolerance/pre-T2DM cases in humans and can be used to uncover potential insights into reduced bone formation as a mechanism of skeletal fragility in these patients.

Associations of Osteocalcin Forms With Metabolic Syndrome and Its Individual Components in Older Men: The Health In Men Study.

CONTEXT: The osteoblast-derived polypeptide, osteocalcin (OC), has been associated with lower risk of type 2 diabetes and metabolic syndrome (MetS) in several epidemiological studies. Animal studies have indicated the undercarboxylated form of OC (ucOC) drives its association with metabolic outcomes. OBJECTIVE: We compared associations of ucOC and carboxylated OC (cOC) with MetS and its components in older men. METHODS: A cross-sectional analysis of 2575 men aged ≥70 years and older resident in Perth, Western Australia. ucOC was assayed using a hydroxyapatite-binding method, and cOC calculated by subtracting ucOC from total OC. Main outcome measures were MetS and its components. RESULTS: Both lower serum ucOC and cOC levels, and the proportion of cOC (%cOC) were associated with less favorable metabolic parameters (higher waist circumference, triglyceride, glucose, blood pressure, and lower high-density lipoprotein cholesterol), whereas inverse associations were found with %ucOC. Men in the lowest quintile of ucOC had higher risk of MetS compared to men in the highest quintile (Q1 ≤ 7.7 vs Q5 > 13.8 ng/mL; OR = 2.4; 95% CI, 1.8-3.2). Men in the lowest quintile of cOC had higher risk of MetS compared to those in the highest quintile (≤ 5.8 vs > 13.0 ng/mL; OR = 2.4; 95% CI, 1.8-3.2). CONCLUSION: Lower concentrations of serum ucOC or cOC were associated with less favorable metabolic parameters and a higher risk of MetS. In contrast, a lower proportion of ucOC was associated with better metabolic parameters and lower MetS risk. Further research is warranted to determine whether ucOC and cOC are suitable biomarkers for cardiometabolic risk in men.

Roles of bone-derived hormones in type 2 diabetes and cardiovascular pathophysiology.

BACKGROUND: Emerging evidence demonstrates that bone is an endocrine organ capable of influencing multiple physiological and pathological processes through the secretion of hormones. Recent research suggests complex crosstalk between the bone and other metabolic and cardiovascular tissues. It was uncovered that three of these bone-derived hormones-osteocalcin, lipocalin 2, and sclerostin-are involved in the endocrine regulations of cardiometabolic health and play vital roles in the pathophysiological process of developing cardiometabolic syndromes such as type 2 diabetes and cardiovascular disease. Chronic low-grade inflammation is one of the hallmarks of cardiometabolic diseases and a major contributor to disease progression. Novel evidence also implicates important roles of bone-derived hormones in the regulation of chronic inflammation. SCOPE OF REVIEW: In this review, we provide a detailed overview of the physiological and pathological roles of osteocalcin, lipocalin 2, and sclerostin in cardiometabolic health regulation and disease development, with a focus on the modulation of chronic inflammation. MAJOR CONCLUSIONS: Evidence supports that osteocalcin has a protective role in cardiometabolic health, and an increase of lipocalin 2 contributes to the development of cardiometabolic diseases partly via pro-inflammatory effects. The roles of sclerostin appear to be complicated: It exerts pro-adiposity and pro-insulin resistance effects in type 2 diabetes and has an anti-calcification effect during cardiovascular disease. A better understanding of the actions of these bone-derived hormones in the pathophysiology of cardiometabolic diseases will provide crucial insights to help further research develop new therapeutic strategies to treat these diseases.

The potential actions of angiotensin-converting enzyme II (ACE2) activator diminazene aceturate (DIZE) in various diseases.

The renin angiotensin system (RAS) regulates fluid balance, blood pressure and maintains vascular tone. The potent vasoconstrictor angiotensin II (Ang II) produced by angiotensin-converting enzyme (ACE) comprises the classical RAS. The non-classical RAS involves the conversion of Ang II via ACE2 into the vasodilator Ang (1-7) to counterbalance the effects of Ang II. Furthermore, ACE2 converts AngA into another vasodilator named alamandine. The over activation of the classical RAS (increased vasoconstriction) and depletion of the non-classical RAS (decreased vasodilation) results in vascular dysfunction. Vascular dysfunction is the leading cause of atherosclerosis and cardiovascular disease (CVD). Additionally, local RAS is expressed in various tissues and regulates cellular functions. RAS dysregulation is involved in other several diseases such as inflammation, renal dysfunction and even cancer growth. An approach in restoring vascular dysfunction and other pathological diseases is to either increase the activity of ACE2 or reduce the effect of the classical RAS by counterbalancing Ang II effects. The antitrypanosomal agent, diminazene aceturate (DIZE), is one approach in activating ACE2. DIZE has been shown to exert beneficial effects in CVD experimental models of hypertension, myocardial infarction, type 1 diabetes and atherosclerosis. Thus, this review focuses on DIZE and its effect in several tissues such as blood vessels, cardiac, renal, immune and cancer cells.

The Effect of Recombinant Undercarboxylated Osteocalcin on Endothelial Dysfunction.

Low circulating levels of undercarboxylated osteocalcin (ucOC) is associated with a higher risk of cardiovascular disease, yet whether ucOC has a direct effect on endothelium-dependent vasorelaxation, or in proximity to its postulated receptor, the class CG protein-coupled receptor (GPCR6A), in blood vessels remains unclear. Immunohistochemistry and proximity ligation assays were used to localize the presence of ucOC and GPRC6A and to determine the physical proximity (< 40 nm) in radial artery segments collected from patients undergoing coronary artery bypass surgery (n = 6) which exhibited calcification (determined by Von Kossa) and aorta from New Zealand white rabbits exhibiting atherosclerotic plaques. Endothelium-dependent vasorelaxation was assessed using cumulative doses of acetylcholine in vitro on abdominal aorta of rabbits fed a normal chow diet (n = 10) and a 4-week atherogenic diet (n = 9) pre-incubated with ucOC (10 ng/mL) or vehicle. Both ucOC and GPRC6A were localized in human and rabbit diseased-blood vessels. Proximity ligation assay staining demonstrated physical proximity of ucOC with GPRC6A only within plaques in rabbit arteries and the endothelium layer of rabbit arterioles. Endothelium-dependent vasorelaxation was impaired in atherogenic abdominal aorta compared to healthy aorta and ucOC attenuated this impairment. ucOC attenuated impaired endothelium-dependent vasorelaxation in rabbit abdominal aorta following an atherogenic diet, however, this effect may be independent of GPRC6A. It is important that future studies determine the underlying cellular mechanisms by which ucOC effects blood vessels as well as whether it can be used as a therapeutic agent against the progression of atherosclerosis.

The effectiveness of high intensity intermittent training on metabolic, reproductive and mental health in women with polycystic ovary syndrome: study protocol for the iHIT- randomised controlled trial.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a reproductive-metabolic condition. Insulin resistance is a hallmark of PCOS and is related to increased hyperandrogenism that drives inherent metabolic, reproductive and psychological features of the syndrome. Insulin resistance in women with PCOS is managed by weight loss, lifestyle interventions (i.e. exercise, diet) and insulin-sensitising medications. This manuscript describes the protocol of our study evaluating the effectiveness of high intensity intermittent training (HIIT) or moderate intensity exercise on cardiometabolic, reproductive and mental health in overweight women with PCOS. METHODS/DESIGN: We will employ a three arm, parallel-group, randomised controlled trial recruiting 60 women diagnosed with PCOS, aged between 18 and 45 years and with a body mass index (BMI) greater than 25 kg/m2. Following screening and baseline testing, women will be randomised by simple randomisation procedure using computer generated sequence allocation to undergo one of two 12-week supervised interventions: either HIIT or moderate intensity exercise (standard supervised exercise), or to standard care [Con] (unsupervised lifestyle advice) at a 1:1:1 allocation ratio. The primary outcome for this trial is to measure the improvements in metabolic health; specifically changes in insulin sensitivity in response to different exercise intensities. Baseline and post-intervention testing include anthropometric measurements, cardiorespiratory fitness testing, reproductive hormone profiles (anti-müllerian hormone and steroid profiles), metabolic health, health-related quality of life and mental health questionnaires and objective and subjective lifestyle monitoring. Reporting of the study will follow the CONSORT statement. DISCUSSION: This trial aims to demonstrate the comparative efficacy and maintenance of different exercise intensities to advance the understanding of PCOS management and provide insight into the optimal exercise intensity for improved cardiometabolic outcomes. Secondary outcomes will include the impact of different exercise protocols on reproductive hormone profiles, mental health and health-related quality of life. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12615000242527 . Registered on 17 March 2015.

Undercarboxylated Osteocalcin Improves Insulin-Stimulated Glucose Uptake in Muscles of Corticosterone-Treated Mice.

Short-term administration of glucocorticoids (GCs) impairs muscle insulin sensitivity at least in part via the reduction of undercarboxylated osteocalcin (ucOC). However, whether ucOC treatment reverses the GC-induced muscle insulin resistance remains unclear. To test the hypothesis that ucOC directly ameliorates impaired insulin-stimulated glucose uptake (ISGU) induced by short-term GC administration in mice muscle and to identify the molecular mechanisms, mice were implanted with placebo or corticosterone (CS) slow-release pellets. Two days post-surgery, insulin-tolerance tests (ITTs) were performed. On day 3, serum was collected and extensor digitorum longus (EDL) and soleus muscles were isolated and treated ex vivo with vehicle, ucOC (30 ng/mL), insulin (60 µU/mL), or both. Circulating hormone levels, muscle glucose uptake, and muscle signaling proteins were assessed. CS administration reduced both serum osteocalcin and ucOC levels, whole-body insulin sensitivity, and muscle ISGU in EDL. Ex vivo ucOC treatment restored ISGU in CS-affected muscle, without increasing non-insulin-stimulated glucose uptake. In CS-affected EDL muscle, ucOC enhanced insulin action on phosphorylated (p-)protein kinase B (Akt)Ser473 and the p-extracellular signal-regulated kinase isoform 2 (ERK2)Thr202/Tyr204 /total (t)ERK2 ratio, which correlated with ISGU. In CS-affected soleus muscle, ucOC enhanced insulin action on p-mammalian target of rapamycin (mTOR)Ser2481 , the p-mTORSer2481 /tmTOR ratio, p-Akt substrate of 160kD (AS160)Thr642 , and p-protein kinase C (PKC) (pan)Thr410 , which correlated with ISGU. Furthermore, p-PKC (pan)Thr410 correlated with p-AktSer473 and p-AS160Thr642 . ucOC exerts direct insulin-sensitizing effects on CS-affected mouse muscle, likely through an enhancement in activity of key proteins involved in both insulin and ucOC signaling pathways. Furthermore, these effects are muscle type-dependent. © 2019 American Society for Bone and Mineral Research.

Potential Role for Osteocalcin in the Development of Atherosclerosis and Blood Vessel Disease.

There is increasing evidence for the involvement of the skeleton in the regulation of atherosclerotic vascular disease. Osteocalcin, an osteoblast derived protein, exists in two forms, carboxylated and undercarboxylated osteocalcin. Undercarboxylated osteocalcin has been linked to the regulation of metabolic functions, including glucose and lipid metabolism. Features of atherosclerosis have been associated with circulating osteocalcin; however, this association is often conflicting and unclear. Therefore, the aim of this review is to examine the evidence for a role of osteocalcin in atherosclerosis development and progression, and in particular endothelial dysfunction and vascular calcification. The current literature suggests that undercarboxylated osteocalcin stimulates the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway to upregulate nitric oxide and nuclear factor kappa ß (NF-кß) in vascular cells, possibly protecting endothelial function and preventing atherogenesis. However, this effect may be mediated by metabolic factors, such as improvements in insulin signaling, rather than through a direct effect on the vasculature. Total osteocalcin is frequently associated with vascular calcification, an association that may occur as a result of vascular cells eliciting an osteogenic phenotype. Whether osteocalcin acts as a mediator or a marker of vascular calcification is currently unclear. As such, further studies that examine each form of osteocalcin are required to elucidate if it is a mediator of atherogenesis, and whether it functions independently of metabolic factors.

Effects of Dietary Inorganic Nitrate Supplementation on Exercise Performance in Patients With Heart Failure: Protocol for a Randomized, Placebo-Controlled, Cross-Over Trial.

BACKGROUND: Chronic heart failure is characterized by an inability of the heart to pump enough blood to meet the demands of the body, resulting in the hallmark symptom of exercise intolerance. Chronic underperfusion of the peripheral tissues and impaired nitric oxide bioavailability have been implicated as contributors to the decrease in exercise capacity in these patients. nitric oxide bioavailability has been identified as an important mediator of exercise tolerance in healthy individuals, but there are limited studies examining the effects in patients with chronic heart failure. OBJECTIVE: The proposed trial is designed to determine the effects of chronic inorganic nitrate supplementation on exercise tolerance in both patients with heart failure preserved ejection fraction (HFpEF) and heart failure reduced ejection fraction (HFrEF) and to determine whether there are any differential responses between the 2 cohorts. A secondary objective is to provide mechanistic insights into the 2 heart failure groups' exercise responses to the nitrate supplementation. METHODS: Patients with chronic heart failure (15=HFpEF and 15=HFrEF) aged 40 to 85 years will be recruited. Following an initial screen cardiopulmonary exercise test, participants will be randomly allocated in a double-blind fashion to consume either a nitrate-rich beetroot juice (16 mmol nitrate/day) or a nitrate-depleted placebo (for 5 days). Participants will continue daily dosing until the completion of the 4 testing visits (maximal cardiopulmonary exercise test, submaximal exercise test with echocardiography, vascular function assessment, and vastus lateralis muscle biopsy). There will then be a 2-week washout period after which the participants will cross over to the other treatment and complete the same 4 testing visits. RESULTS: This study is funded by National Heart Foundation of Australia and Victoria University. Enrolment has commenced and the data collection is expected to be completed in mid 2018. The initial results are expected to be submitted for publication by the end of 2018. CONCLUSIONS: If inorganic nitrate supplementation can improve exercise tolerance in patients with chronic heart failure, it has the potential to aid in further refining the treatment of patients in this population. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12615000906550; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=368912 (Archived by WebCite at http://www.webcitation.org/6xymLMiFK).

Exercise Professionals with Advanced Clinical Training Should be Afforded Greater Responsibility in Pre-Participation Exercise Screening: A New Collaborative Model between Exercise Professionals and Physicians.

Regular exercise improves health but can also induce adverse responses. Although such episodes are rare, many guidelines for pre-participation exercise screening have historically had a low threshold for recommending medical clearance prior to the commencement of exercise, placing the responsibility for decision making about exercise participation on physicians. The 'clearance to exercise' model still occurs widely in practice, but creates cost burdens and barriers to the uptake of exercise. Moreover, many physicians are not provided the training, nor time in a standard consultation, to be able to effectively perform this role. We present a model for pre-participation exercise screening and the initial assessment of clients wishing to commence an exercise programme. It is designed to guide professional practice for the referral, assessment and prescription of exercise for people across the health spectrum, from individuals who are apparently healthy, through to clients with pre-existing or occult chronic conditions. The model removes the request that physicians provide a 'clearance' for patients to engage in exercise programmes. Instead the role of physicians is identified as providing relevant clinical guidance to suitably qualified exercise professionals to allow them to use their knowledge, skills and expertise in exercise prescription to assess and manage any risks related to the prescription and delivery of appropriate exercise programmes. It is anticipated that removing unjustified barriers to exercise participation, such as mandated medical review, will improve the uptake of exercise by the unacceptably high proportion of the population who do not undertake sufficient physical activity for health benefit.

Uncarboxylated Osteocalcin Enhances Glucose Uptake Ex Vivo in Insulin-Stimulated Mouse Oxidative But Not Glycolytic Muscle.

Uncarboxylated osteocalcin (ucOC) stimulates muscle glucose uptake in mice EDL and soleus muscles. However, whether ucOC also exerts a similar effect in insulin-stimulated muscles in a muscle type-specific manner is currently unclear. We aimed to test the hypothesis that, with insulin stimulation, ucOC per se has a greater effect on oxidative muscle compared with glycolytic muscle, and to explore the underlying mechanisms. Mouse (C57BL6, male 9-12 weeks) extensor digitorum longus (EDL) and soleus muscles were isolated and longitudinally split into halves. Muscle samples were treated with varying doses of recombinant ucOC (0, 0.3, 1, 3, 30 ng/ml), followed by insulin addition. Muscle glucose uptake, protein phosphorylation and total expression of protein kinase B (Akt), Akt substrate of 160 kDa (AS160), extracellular signal-regulated kinase isoform 2 (ERK2), and adenosine monophosphate-activated protein kinase subunit α (AMPKα) were assessed. ucOC treatment at 30 ng/ml enhanced muscle glucose uptake in insulin-stimulated soleus, a mainly oxidative muscle (17.5%, p < 0.05), but not in EDL-a mostly glycolytic muscle. In insulin-stimulated soleus only, ucOC treatment (3 and 30 ng/ml) increased phosphorylation of AS160 and ERK2, but not Akt or AMPKα. The ucOC-induced increase in ERK2 phosphorylation in soleus was not associated with the increase in glucose uptake or AS160 phosphorylation. ucOC enhances glucose uptake and AS160 phosphorylation in insulin-stimulated oxidative but not glycolytic muscle, via upstream mechanisms which appear to be independent of ERK or AMPK.

Recombinant Uncarboxylated Osteocalcin Per Se Enhances Mouse Skeletal Muscle Glucose Uptake in both Extensor Digitorum Longus and Soleus Muscles.

Emerging evidence suggests that undercarboxylated osteocalcin (ucOC) improves muscle glucose uptake in rodents. However, whether ucOC can directly increase glucose uptake in both glycolytic and oxidative muscles and the possible mechanisms of action still need further exploration. We tested the hypothesis that ucOC per se stimulates muscle glucose uptake via extracellular signal-regulated kinase (ERK), adenosine monophosphate-activated protein kinase (AMPK), and/or the mechanistic target of rapamycin complex 2 (mTORC2)-protein kinase B (AKT)-AKT substrate of 160 kDa (AS160) signaling cascade. Extensor digitorum longus (EDL) and soleus muscles from male C57BL/6 mice were isolated, divided into halves, and then incubated with ucOC with or without the pretreatment of ERK inhibitor U0126. ucOC increased muscle glucose uptake in both EDL and soleus. It also enhanced phosphorylation of ERK2 (Thr202/Tyr204) and AS160 (Thr642) in both muscle types and increased mTOR phosphorylation (Ser2481) in EDL only. ucOC had no significant effect on the phosphorylation of AMPKα (Thr172). The inhibition of ucOC-induced ERK phosphorylation had limited effect on ucOC-stimulated glucose uptake and AS160 phosphorylation in both muscle types, but appeared to inhibit the elevation in AKT phosphorylation only in EDL. Taken together, ucOC at the physiological range directly increased glucose uptake in both EDL and soleus muscles in mouse. The molecular mechanisms behind this ucOC effect on muscle glucose uptake seem to be muscle type-specific, involving enhanced phosphorylation of AS160 but limitedly modulated by ERK phosphorylation. Our study suggests that, since ucOC increases muscle glucose uptake without insulin, it could be considered as a potential agent to improve muscle glucose uptake in insulin resistant conditions.