Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells.

BACKGROUND: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. METHODS: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. RESULTS: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. CONCLUSIONS: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10-12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.

Growth of human breast tissues from patient cells in 3D hydrogel scaffolds.

BACKGROUND: Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. METHODS: Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. RESULTS: When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix. CONCLUSIONS: These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.

Gastric Cancer Detection with Ensemble Learning on Digital Pathology: Use Case of Gastric Cancer on GasHisSDB Dataset.

Gastric cancer has become a serious worldwide health concern, emphasizing the crucial importance of early diagnosis measures to improve patient outcomes. While traditional histological image analysis is regarded as the clinical gold standard, it is labour intensive and manual. In recognition of this problem, there has been a rise in interest in the use of computer-aided diagnostic tools to help pathologists with their diagnostic efforts. In particular, deep learning (DL) has emerged as a promising solution in this sector. However, current DL models are still restricted in their ability to extract extensive visual characteristics for correct categorization. To address this limitation, this study proposes the use of ensemble models, which incorporate the capabilities of several deep-learning architectures and use aggregate knowledge of many models to improve classification performance, allowing for more accurate and efficient gastric cancer detection. To determine how well these proposed models performed, this study compared them with other works, all of which were based on the Gastric Histopathology Sub-Size Images Database, a publicly available dataset for gastric cancer. This research demonstrates that the ensemble models achieved a high detection accuracy across all sub-databases, with an average accuracy exceeding 99%. Specifically, ResNet50, VGGNet, and ResNet34 performed better than EfficientNet and VitNet. For the 80 × 80-pixel sub-database, ResNet34 exhibited an accuracy of approximately 93%, VGGNet achieved 94%, and the ensemble model excelled with 99%. In the 120 × 120-pixel sub-database, the ensemble model showed 99% accuracy, VGGNet 97%, and ResNet50 approximately 97%. For the 160 × 160-pixel sub-database, the ensemble model again achieved 99% accuracy, VGGNet 98%, ResNet50 98%, and EfficientNet 92%, highlighting the ensemble model's superior performance across all resolutions. Overall, the ensemble model consistently provided an accuracy of 99% across the three sub-pixel categories. These findings show that ensemble models may successfully detect critical characteristics from smaller patches and achieve high performance. The findings will help pathologists diagnose gastric cancer using histopathological images, leading to earlier identification and higher patient survival rates.

Multi-Scale Digital Pathology Patch-Level Prostate Cancer Grading Using Deep Learning: Use Case Evaluation of DiagSet Dataset.

Prostate cancer remains a prevalent health concern, emphasizing the critical need for early diagnosis and precise treatment strategies to mitigate mortality rates. The accurate prediction of cancer grade is paramount for timely interventions. This paper introduces an approach to prostate cancer grading, framing it as a classification problem. Leveraging ResNet models on multi-scale patch-level digital pathology and the Diagset dataset, the proposed method demonstrates notable success, achieving an accuracy of 0.999 in identifying clinically significant prostate cancer. The study contributes to the evolving landscape of cancer diagnostics, offering a promising avenue for improved grading accuracy and, consequently, more effective treatment planning. By integrating innovative deep learning techniques with comprehensive datasets, our approach represents a step forward in the pursuit of personalized and targeted cancer care.

Ensemble Deep Learning-Based Image Classification for Breast Cancer Subtype and Invasiveness Diagnosis from Whole Slide Image Histopathology.

Cancer diagnosis and classification are pivotal for effective patient management and treatment planning. In this study, a comprehensive approach is presented utilizing ensemble deep learning techniques to analyze breast cancer histopathology images. Our datasets were based on two widely employed datasets from different centers for two different tasks: BACH and BreakHis. Within the BACH dataset, a proposed ensemble strategy was employed, incorporating VGG16 and ResNet50 architectures to achieve precise classification of breast cancer histopathology images. Introducing a novel image patching technique to preprocess a high-resolution image facilitated a focused analysis of localized regions of interest. The annotated BACH dataset encompassed 400 WSIs across four distinct classes: Normal, Benign, In Situ Carcinoma, and Invasive Carcinoma. In addition, the proposed ensemble was used on the BreakHis dataset, utilizing VGG16, ResNet34, and ResNet50 models to classify microscopic images into eight distinct categories (four benign and four malignant). For both datasets, a five-fold cross-validation approach was employed for rigorous training and testing. Preliminary experimental results indicated a patch classification accuracy of 95.31% (for the BACH dataset) and WSI image classification accuracy of 98.43% (BreakHis). This research significantly contributes to ongoing endeavors in harnessing artificial intelligence to advance breast cancer diagnosis, potentially fostering improved patient outcomes and alleviating healthcare burdens.

Inhibition of Pref-1 (preadipocyte factor 1) by oestradiol in adolescent girls with anorexia nervosa is associated with improvement in lumbar bone mineral density.

OBJECTIVE: Adolescents with anorexia nervosa (AN) are amenorrheic and have decreased bone mass accrual and low bone mineral density (BMD). The regulation of mesenchymal stem cell differentiation is an important factor governing bone formation. Preadipocyte factor 1 (Pref-1), an inhibitor of adipocyte and osteoblast differentiation, is elevated in states of oestrogen deficiency. In this study, we aim to (i) investigate effects of transdermal oestradiol on Pref-1 in adolescent girls with AN, and (ii) examine associations of changes in Pref-1 with changes in lumbar BMD and bone turnover markers. DESIGN: Adolescent girls with AN and normal-weight controls were studied cross-sectionally. Girls with AN were examined longitudinally in a double-blind study and received transdermal oestradiol (plus cyclic medroxyprogesterone) or placebo for 12 months. PATIENTS: Sixty-nine girls (44 with AN, 25 normal-weight controls) 13-18 years were studied at baseline; 22 AN girls were followed prospectively. MEASUREMENTS: Pref-1 levels, bone formation and resorption markers, and BMD. RESULTS: Pref-1 levels decreased in girls with AN after treatment with transdermal oestradiol compared with placebo (-0·015 ± 0·016 vs 0·060±0·026 ng/ml, P = 0·01), although at baseline, levels did not differ in AN vs controls (0·246 ± 0·015 vs 0·267 ± 0·022 ng/ml). Changes in Pref-1 over 12 months correlated inversely with changes in lumbar BMD (r = -0·48, P = 0·02) and positively with changes in CTX (r = 0·73, P = 0·006). CONCLUSIONS: For the first time, we show that Pref-1 is negatively regulated by oestradiol in adolescent girls with AN. Inhibition of Pref-1 may mediate the beneficial effects of transdermal oestradiol replacement on BMD in girls with AN.

Enhancing Prostate Cancer Diagnosis with a Novel Artificial Intelligence-Based Web Application: Synergizing Deep Learning Models, Multimodal Data, and Insights from Usability Study with Pathologists.

Prostate cancer remains a significant cause of male cancer mortality in the United States, with an estimated 288,300 new cases in 2023. Accurate grading of prostate cancer is crucial for ascertaining disease severity and shaping treatment strategies. Modern deep learning techniques show promise in grading biopsies, but there is a gap in integrating these advances into clinical practice. Our web platform tackles this challenge by integrating human expertise with AI-driven grading, incorporating diverse data sources. We gathered feedback from four pathologists and one medical practitioner to assess usability and real-world alignment through a survey and the NASA TLX Usability Test. Notably, 60% of users found it easy to navigate, rating it 5.5 out of 7 for ease of understanding. Users appreciated self-explanatory information in popup tabs. For ease of use, all users favored the detailed summary tab, rating it 6.5 out of 7. While 80% felt patient demographics beyond age were unnecessary, high-resolution biopsy images were deemed vital. Acceptability was high, with all users willing to adopt the app, and some believed it could reduce workload. The NASA TLX Usability Test indicated a low-moderate perceived workload, suggesting room for improved explanations and data visualization.

The heparin-binding domain of IGFBP-2 has insulin-like growth factor binding-independent biologic activity in the growing skeleton.

Insulin-like growth factor-binding protein 2 (IGFBP-2) is a member of a family of six highly conserved IGFBPs that are carriers for the insulin-like growth factors (IGFs). IGFBP-2 levels rise during rapid neonatal growth and at the time of peak bone acquisition. In contrast, Igfbp2(-/-) mice have low bone mass accompanied by reduced osteoblast numbers, low bone formation rates, and increased PTEN expression. In the current study, we postulated that IGFBP-2 increased bone mass partly through the activity of its heparin-binding domain (HBD). We synthesized a HBD peptide specific for IGFBP-2 and demonstrated in vitro that it rescued the mineralization phenotype of Igfbp2(-/-) bone marrow stromal cells and calvarial osteoblasts. Consistent with its cellular actions, the HBD peptide ex vivo stimulated metacarpal periosteal expansion. Furthermore, administration of HBD peptide to Igfbp2(-/-) mice increased osteoblast number, suppressed marrow adipogenesis, restored trabecular bone mass, and reduced bone resorption. Skeletal rescue in the Igfbp2(-/-) mice was characterized by reduced PTEN expression followed by enhanced Akt phosphorylation in response to IGF-I and increased ß-catenin signaling through two mechanisms: 1) stimulation of its cytosolic accumulation and 2) increased phosphorylation of serine 552. We conclude that the HBD peptide of IGFBP-2 has anabolic activity by activating IGF-I/Akt and ß-catenin signaling pathways. These data support a growing body of evidence that IGFBP-2 is not just a transport protein but rather that it functions coordinately with IGF-I to stimulate growth and skeletal acquisition.

Multiscale anisotropy analysis of second-harmonic generation collagen imaging of human pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a minority (< 10%) of patients surviving five years past diagnosis. This could be improved with the development of new imaging modalities for early differentiation of benign and cancerous fibrosis. This study intends to explore the application of a two-photon microscopy technique known as second harmonic generation to PDAC using the 2D Wavelet Transform Modulus Maxima (WTMM) Anisotropy method to quantify collagen organization in fibrotic pancreatic tissue. Forty slides from PDAC patients were obtained and eight images were captured per each tissue category on each slide. Brownian surface motion and white noise images were generated for calibration and testing of a new variable binning approach to the 2D WTMM Anisotropy method. The variable binning method had greater resistance to wavelet scaling effects and white noise images were found to have the lowest anisotropy factor. Cancer and fibrosis had greater anisotropy factors (Fa) at small wavelet scales than normal and normal adjacent tissue. At a larger scale of 21 µm this relationship changed with normal tissue having a higher Fa than all other tissue groups. White noise is the best representative image for isotropy and the 2D WTMM anisotropy method is sensitive to changes induced in collagen by PDAC.

Growth hormone is positively associated with surrogate markers of bone turnover during puberty.

BACKGROUND: Puberty is characterized by increases in growth hormone (GH) and insulin-like growth factor-1 (IGF-1) and the pubertal growth spurt. Bone formation and resorption also increase, consistent with increased bone metabolism. OBJECTIVE: To determine the relationship between pubertal bone metabolism, GH and IGF-1. We hypothesized that bone turnover peaks at the time of greatest pubertal GH secretion. DESIGN AND SUBJECTS: Subjects included 86 girls and boys, 9-17 years-old (BMI 10th-90th percentiles). Because higher endogenous GH secretion is associated with a higher nadir following oral glucose, we used the GH nadir following a 2-h OGTT as indicative of GH status. Fasting serum IGF-1, aminoterminal propeptide of type 1 procollagen (P1NP) and carboxy-terminal collagen crosslinks (CTX) were obtained. Subjects were grouped per expected timing of peak growth. Group 1: Tanner 1 girls and Tanner 1-2 boys (period preceding peak growth), Group 2: Tanner 2-3 girls and Tanner 3-4 boys (period of peak growth) and Group 3: Tanner 4-5 girls and Tanner 5 boys (period following peak growth). RESULTS: GH peaked at mid-puberty (Group 2) and IGF-1 in late puberty (Group 3). P1NP and CTX were highest in mid-puberty compared with early and late puberty (P = 0·0009 and 0·006 in girls and P = 0·005 and 0·04 in boys). GH, but not IGF-1, correlated with P1NP (r = 0·46 in both genders, P ≤ 0·008) and CTX (r = 0·37 and 0·38, P = 0·04 and 0·02 in girls and boys, respectively). Similarly, on regression modelling, GH (but not IGF-1) predicted both bone turnover markers in both genders. CONCLUSION: GH is strongly associated with pubertal bone metabolism, independent of systemic IGF-1 in girls and boys.

Treatment With a Ghrelin Agonist in Outpatient Women With Anorexia Nervosa: A Randomized Clinical Trial.

OBJECTIVE: To assess the effects of relamorelin-an agonist of the appetite-stimulating hormone ghrelin, which has effects on gastric emptying-on (1) weight gain and (2) gastric emptying in women with anorexia nervosa. METHODS: In a randomized, double-blind, placebo-controlled design, the effects of the ghrelin agonist relamorelin were studied in 22 outpatient women with anorexia nervosa, diagnosed using DSM-5 criteria. The study was conducted at the Massachusetts General Hospital Clinical Research Center between March 11, 2013, and February 26, 2015. Ten participants were randomly assigned to relamorelin 100 µg subcutaneously daily (mean ± SEM age: 28.9 ± 2.4 y), and 12 were randomly assigned to placebo (28.9 ± 1.9 y). We measured changes in weight and gastric emptying time using a gastric emptying breath test (GEBT) for relamorelin versus placebo after 4 weeks of treatment. RESULTS: At baseline, subjects did not differ in weight, plasma ghrelin levels, or gastric emptying time. Three subjects randomized to relamorelin stopped use of the study medication due to reported feelings of increased hunger. After 4 weeks, there was a trend toward an increase in weight in participants randomized to relamorelin (mean ± SEM change: 0.86 ± 0.40 kg) compared to placebo (0.04 ± 0.28 kg; P = .07), and gastric emptying time was significantly shorter in patients taking relamorelin (median [interquartile range]: 58.0 [51.0, 78.0] minutes) compared to placebo (85.0 [75.8,100.5] minutes; P = .03). CONCLUSIONS: Treatment with a ghrelin agonist in women with anorexia nervosa significantly decreases gastric emptying time, leads to a trend in weight gain after only 4 weeks, and is well-tolerated. Further study is necessary to determine the long-term safety and efficacy of a ghrelin agonist in the treatment of anorexia nervosa. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01642550.

Effects of growth hormone administration for 6 months on bone turnover and bone marrow fat in obese premenopausal women.

PURPOSE: Abdominal adiposity is associated with low BMD and decreased growth hormone (GH) secretion, an important regulator of bone homeostasis. The purpose of our study was to determine the effects of a short course of GH on markers of bone turnover and bone marrow fat in premenopausal women with abdominal adiposity. MATERIALS AND METHODS: In a 6-month, randomized, double-blind, placebo-controlled trial we studied 79 abdominally obese premenopausal women (21-45 y) who underwent daily sc injections of GH vs. placebo. Main outcome measures were body composition by DXA and CT, bone marrow fat by proton MR spectroscopy, P1NP, CTX, 25(OH)D, hsCRP, undercarboxylated osteocalcin (ucOC), preadipocyte factor 1 (Pref 1), apolipoprotein B (ApoB), and IGF-1. RESULTS: GH increased IGF-1, P1NP, 25(OH)D, ucOC, bone marrow fat and lean mass, and decreased abdominal fat, hsCRP, and ApoB compared with placebo (p<0.05). There was a trend toward an increase in CTX and Pref-1. Among all participants, a 6-month increase in IGF-1 correlated with 6-month increase in P1NP (p=0.0005), suggesting that subjects with the greatest increases in IGF-1 experienced the greatest increases in bone formation. A six-month decrease in abdominal fat, hsCRP, and ApoB inversely predicted 6-month change in P1NP, and 6-month increase in lean mass and 25(OH)D positively predicted 6-month change in P1NP (p≤0.05), suggesting that subjects with greatest decreases in abdominal fat, inflammation and ApoB, and the greatest increases in lean mass and 25(OH)D experienced the greatest increases in bone formation. A six-month increase in bone marrow fat correlated with 6-month increase in P1NP (trend), suggesting that subjects with the greatest increases in bone formation experienced the greatest increases in bone marrow fat. Forward stepwise regression analysis indicated that increase in lean mass and decrease in abdominal fat were positive predictors of P1NP. When IGF-1 was added to the model, it became the only predictor of P1NP. CONCLUSION: GH replacement in abdominally obese premenopausal women for 6 months increased bone turnover and bone marrow fat. Reductions in abdominal fat, and inflammation, and increases in IGF-1, lean mass and vitamin D were associated with increased bone formation. The increase in bone marrow fat may reflect changes in energy demand from increased bone turnover.

Attenuating homologous recombination stimulates an AID-induced antileukemic effect.

Activation-induced cytidine deaminase (AID) is critical in normal B cells to initiate somatic hypermutation and immunoglobulin class switch recombination. Accumulating evidence suggests that AID is also prooncogenic, inducing cancer-promoting mutations or chromosome rearrangements. In this context, we find that AID is expressed in >40% of primary human chronic lymphocytic leukemia (CLL) cases, consistent with other reports. Using a combination of human B lymphoid leukemia cells and mouse models, we now show that AID expression can be harnessed for antileukemic effect, after inhibition of the RAD51 homologous recombination (HR) factor with 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS). As a proof of principle, we show that DIDS treatment inhibits repair of AID-initiated DNA breaks, induces apoptosis, and promotes cytotoxicity preferentially in AID-expressing human CLL. This reveals a novel antineoplastic role of AID that can be triggered by inhibition of HR, suggesting a potential new paradigm to treat AID-expressing tumors. Given the growing list of tumor types with aberrant AID expression, this novel therapeutic approach has potential to impact a significant patient population.

Marrow fat and preadipocyte factor-1 levels decrease with recovery in women with anorexia nervosa.

Women with anorexia nervosa (AN) have elevated marrow fat mass despite low visceral and subcutaneous fat depots, which is inversely associated with bone mineral density (BMD). Whether marrow fat mass remains persistently elevated or decreases with recovery from AN is currently unknown. In this study, we investigated changes in marrow fat in women who have recovered from AN (AN-R). We also studied the relationship between preadipocyte factor (Pref)-1-a member of the EGF-like family of proteins and regulator of adipocyte and osteoblast differentiation-and fat depots and BMD in AN-R compared with women with AN and healthy controls (HC). We studied 29 women: 14 with active or recovered AN (30.7 + 2.2 years [mean ± SEM]) and 15 normal-weight controls (27.8 ± 1.2 years). We measured marrow adipose tissue (MAT) of the L4 vertebra and femur by (1) H-magnetic resonance spectroscopy; BMD of the spine, hip, and total body by DXA; and serum Pref-1 and leptin levels. We found that MAT of the L4 vertebra was significantly lower in AN-R compared with AN (p = 0.03) and was comparable to levels in HC. Pref-1 levels were also significantly lower in AN-R compared with AN (p = 0.02) and comparable to levels in healthy controls. Although Pref-1 was positively associated with MAT of the L4 vertebra in AN (R = 0.94; p = 0.002), we found that it was inversely associated with MAT of the L4 vertebra in HC (R = -0.71; p = 0.004). Therefore, we have shown that MAT and Pref-1 levels decrease with recovery from AN. Our data suggest that Pref-1 may have differential effects in states of nutritional deprivation compared with nutritional sufficiency.

Sclerostin levels and bone turnover markers in adolescents with anorexia nervosa and healthy adolescent girls.

Sclerostin, product of the SOST gene, is an important determinant of bone formation and resorption. Adolescents with anorexia nervosa (AN) have low bone density and decreased levels of bone turnover markers. However, sclerostin has not been examined in AN as a potential mediator of impaired bone metabolism. Our study objectives were to (i) assess associations of sclerostin with surrogate bone turnover markers in girls with AN and controls and (ii) examine effects of transdermal estradiol on sclerostin in AN. 69 girls (44 with AN and 25 normal-weight controls) 13-18 years old were studied at baseline. 22 AN girls were randomized to transdermal estradiol (plus cyclic medroxyprogesterone) or placebo in a double-blind study for 12 months. Sclerostin correlated positively with P1NP and CTX in controls (r=0.67 and 0.53, p=0.0002 and 0.005, respectively) but not in AN despite comparable levels at baseline. Changes in sclerostin over twelve months did not differ in girls randomized to estradiol or placebo. The relationship between sclerostin and bone turnover markers is disrupted in adolescent girls with AN. Despite an increase in BMD with estradiol administration in AN, estrogen does not impact sclerostin levels in this group.

Moderate alcohol intake lowers biochemical markers of bone turnover in postmenopausal women.

OBJECTIVE: Epidemiological studies indicate that higher bone mass is associated with moderate alcohol consumption in postmenopausal women. However, the underlying cellular mechanisms responsible for the putative beneficial effects of alcohol on bone are unknown. Excessive bone turnover, combined with an imbalance whereby bone resorption exceeds bone formation, is the principal cause of postmenopausal bone loss. This study investigated the hypothesis that moderate alcohol intake attenuates bone turnover after menopause. METHODS: Bone mineral density was determined by dual-energy x-ray absorptiometry in 40 healthy postmenopausal women (mean ± SE age, 56.3 ± 0.5 y) who consumed alcohol at 19 ± 1 g/day. Serum levels of the bone formation marker osteocalcin and the resorption marker C-terminal telopeptide (CTx) were measured by immunoassay at baseline (day 0) and after alcohol withdrawal for 14 days. Participants then consumed alcohol and were assayed on the following morning. RESULTS: Bone mineral density at the trochanter and total hip were positively correlated to the level of alcohol consumption. Serum osteocalcin and CTx increased after abstinence (4.1 ± 1.6%, P = 0.01 and 5.8 ± 2.6%, P = 0.02 compared with baseline, respectively). Osteocalcin and CTx decreased after alcohol readministration, compared with the previous day (-3.4 ± 1.4%, P = 0.01 and -3.5 ± 2.1%, P = 0.05, respectively), to values that did not differ from baseline (P > 0.05). CONCLUSIONS: Abstinence from alcohol results in increased markers of bone turnover, whereas resumption of alcohol reduces bone turnover markers. These results suggest a cellular mechanism for the increased bone density observed in postmenopausal moderate alcohol consumers. Specifically, the inhibitory effect of alcohol on bone turnover attenuates the detrimental skeletal consequences of excessive bone turnover associated with menopause.

Effects of GH in women with abdominal adiposity: a 6-month randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Abdominal adiposity is associated with increased cardiovascular risk and decreased GH secretion. The objective of our study was to determine the effects of GH on body composition and cardiovascular risk markers in abdominally obese women. MATERIALS AND METHODS: In this randomized, double-blind, placebo-controlled study, 79 obese premenopausal women received GH vs placebo for 6 months. Primary endpoints were i) total abdominal (total abdominal adipose tissue, TAT) fat by computed tomography (CT) (body composition) and ii) high-sensitivity C-reactive protein (hsCRP) (cardiovascular risk marker). Body composition was assessed by CT, dual-energy X-ray absorptiometry, and proton MR spectroscopy. Serum cardiovascular risk markers, carotid intima-media thickness, and endothelial function were measured. RESULTS: Mean 6-month GH dose was 1.7±0.1 mg/day, resulting in a mean IGF1 SDS increase from -1.7±0.08 to -0.1±0.3 in the GH group. GH administration decreased TAT and hsCRP compared with placebo. In addition, it increased thigh muscle mass and lean body mass and decreased subcutaneous abdominal and trunk fat, tissue plasminogen activator, apoB, and apoB/low-density lipoprotein compared with placebo. Visceral adipose tissue (VAT) decreased and intramyocellular lipid increased within the GH group. Six-month change in IGF1 levels was negatively associated with 6-month decrease in TAT and VAT. One subject had a 2 h glucose >200 mg/ml at 3 months; four subjects, three of whom were randomized to GH, had 2 h glucose levels >200 mg/ml at the end of the study. CONCLUSION: GH administration in abdominally obese premenopausal women exerts beneficial effects on body composition and cardiovascular risk markers but is associated with a decrease in glucose tolerance in a minority of women.

Preadipocyte factor-1 levels are higher in women with hypothalamic amenorrhea and are associated with bone mineral content and bone mineral density through a mechanism independent of leptin.

CONTEXT: Preadipocyte factor 1 (pref-1) is increased in anorexia nervosa and is associated negatively with bone mineral density (BMD). No previous studies exist on pref-1 in women with exercise-induced hypothalamic amenorrhea (HA), which similar to anorexia nervosa, is an energy-deficiency state associated with hypoleptinemia. OBJECTIVE: Our objective was to evaluate whether pref-1 levels are also elevated and associated with low BMD and to assess whether leptin regulates pref-1 levels in women with HA. DESIGN: Study 1 was a double-blinded, placebo-controlled randomized clinical trial of metreleptin administration in women with HA. Study 2 was an open-label study of metreleptin administration in low physiological, supraphysiological, and pharmacological doses in healthy women volunteers. SETTING AND PATIENTS: At Beth Israel Deaconess Medical Center, 20 women with HA and leptin levels higher than 5 ng/ml and nine healthy control women participated in study 1, and five healthy women participated in study 2. INTERVENTION: For study 1, 20 HA subjects were randomized to receive either 0.08 mg/kg metreleptin (n = 11) or placebo (n = 9). For study 2, five healthy subjects received 0.01, 0.1, and 0.3 mg/kg metreleptin in both fed and fasting conditions for 1 and 3 d, respectively. MAIN OUTCOME MEASURES: Circulating pref-1 and leptin levels were measured. RESULTS: Pref-1 was significantly higher in HA subjects vs. controls (P = 0.035) and negatively associated with BMD (ρ = -0.38; P < 0.01) and bone mineral content (ρ = -0.32; P < 0.05). Metreleptin administration did not alter pref-1 levels in any study reported herein. CONCLUSIONS: Pref-1 is higher in HA subjects than controls. Metreleptin administration at low physiological, supraphysiological, and pharmacological doses does not affect pref-1 levels, suggesting that hypoleptinemia is not responsible for higher pref-1 levels and that leptin does not regulate pref-1.

Determinants of bone mineral density in obese premenopausal women.

Despite being a risk factor for cardiovascular disease and diabetes mellitus, obesity has been thought to protect against osteoporosis. However, recent studies have demonstrated a differential impact of specific fat compartments on bone mineral density (BMD) with visceral adipose tissue (VAT) having potential detrimental effects on BMD. Visceral obesity is also associated with dysregulation of the GH/IGF-1 axis, an important regulator of bone homeostasis. The purpose of our study was to evaluate the differential effects of abdominal fat depots and muscle, vitamin D, and hormonal determinants, including insulin-like growth factor-1 (IGF-1), testosterone, and estradiol, on trabecular BMD of the lumbar spine. We studied 68 healthy obese premenopausal women (mean BMI, 36.7±4.2 kg/m(2)). Quantitative computed tomography (QCT) was used to assess body composition and lumbar trabecular BMD. There was an inverse association between BMD and VAT, independent of age and BMI (p=0.003). IGF-1 correlated positively with BMD and negatively with VAT and, in stepwise multivariate regression modeling, was the strongest predictor of BMD and procollagen type 1 amino-terminal propeptide (P1NP). Thigh muscle cross sectional area (CSA) and thigh muscle density were also associated with BMD (p<0.05), but 25-hydroxyvitamin D [25(OH)D], testosterone, free testosterone, and estradiol levels were not. 25(OH)D was associated inversely with BMI, total, and subcutaneous abdominal adipose tissue (p<0.05). These findings support the hypothesis that VAT exerts detrimental effects, whereas muscle mass exerts positive effects on BMD in premenopausal obese women. Moreover, our findings suggest that IGF-1 may be a mediator of the deleterious effects of VAT on bone health through effects on bone formation.