Khosla, Sundeep

Abstract Calorie restriction (CR) with adequate nutrient intake is a potential geroprotective intervention. To advance this concept in humans, we tested the hypothesis that moderate CR in healthy young‐to‐middle‐aged individuals would reduce circulating biomarkers of cellular senescence, a fundamental mechanism of aging and aging‐related conditions. Using plasma specimens from the Comprehensive Assessment of Long‐term Effects of Reducing Intake of Energy (CALERIE™) phase 2 study, we found that CR significantly reduced the concentrations of several senescence biomarkers at 12 and 24 months compared to an ad libitum diet. Using machine learning, changes in biomarker concentrations emerged as important predictors of the change in HOMA‐IR and insulin sensitivity index at 12 and 24 months, and the change in resting metabolic rate residual at 12 months. Finally, using adipose tissue RNA‐sequencing data from a subset of participants, we observed a significant reduction in a senescence‐focused gene set in response to CR at both 12 and 24 months compared to baseline. Our results advance the understanding of the effects of CR in humans and further support a link between cellular senescence and metabolic health.

Abstract Disclosure: C.D. Zhang: None. S. Singh: None. J. Saini: None. M. Suresh: None. K. Thangamuthu: None. R. Nathani: None. A.L. Ebbehøj: None. V. Fell: None. S.J. Achenbach: None. E.J. Atkinson: None. S. Khosla: None. I. Bancos: None. Background: Patients with mild autonomous cortisol secretion (MACS) have higher rates of fractures compared to patients with non-functioning adrenal nodules, but bone mass is typically only modestly reduced. We hypothesized that chronic exposure to mild levels of cortisol excess may lead to a deterioration in bone quality. Objective: To characterize the effect of MACS on bone metabolism and bone microarchitecture, as measured by high-resolution peripheral quantitative computed tomography (HR-pQCT). Methods: In this cross-sectional single-center study, patients with MACS were matched by age, sex, and gonadal status to referent subjects without adrenal nodules. MACS was defined as serum cortisol concentration >1.8 mcg/dL after the 1 mg overnight dexamethasone suppression test (1-mg DST), in the absence of a classic Cushingoid phenotype. Those with other known secondary causes of osteoporosis and/or on pharmacologic treatment for low bone mass were excluded. All study participants underwent a bone health interview, blood draw for bone turnover markers, dual-energy x-ray absorptiometry (DXA) scan with trabecular bone score (TBS), and HR-pQCT of the non-dominant radius and tibia. Participants with a recent fracture within 12 months were excluded from the analysis of bone turnover markers. The Kruskal-Wallis test was used to compare measurements between groups. Results: A total of 82 patients with MACS and 82 referent subjects were prospectively enrolled between 1/2019 and 7/2022. The median age of study participants at the time of enrolment was 57 years (interquartile range [IQR], 47-65), and 57 (70%) were women. In the MACS cohort, the median cortisol following 1-mg DST was 3.2 mcg/dL (IQR, 2.3-6.5) with median ACTH of 8.7 pg/mL (IQR, 6.0-14.0) and median DHEA-S of 42.0 mcg/dL (IQR 23.0-68.0). While no differences in bone mineral density (BMD) were observed at any site (lumbar spine, total hip, femoral neck, or distal radius) between the two groups, patients with MACS had decreased TBS (1.374 vs 1.467, P <0.001) and lower osteocalcin concentrations (16.9 vs. 19.7 ng/mL, P = 0.005) than referent subjects without adrenal nodules. On HR-pQCT at the tibia, patients with MACS had significantly reduced trabecular volumetric BMD (151.6 vs 167.4 mg hydroxyapatite/cm3, P = 0.002), trabecular bone volume/tissue volume ratio (0.219 vs 0.237, P = 0.011), and number of trabeculae (1.428 vs 1.506 1/mm, P = 0.045), while trabecular separation (0.707 vs 0.656, P = 0.023) was increased compared to referent subjects. Conclusions: Patients with MACS have altered bone metabolism and microstructural deterioration that is not evident on routine DXA BMD testing. These changes may mediate their increased fracture risk and could serve as biomarkers of cortisol-induced changes in the bone. Presentation: Thursday, June 15, 2023

Abstract Disclosure: J. Saini: None. R. Nathani: None. S. Singh: None. V. Fell: None. E. Atkinson: None. S. Achenbach: None. S. Khosla: None. I. Bancos: None. Background: Previous studies reported that both cortisol secretion and bone metabolism biomarkers follow a circadian pattern of secretion. Patients with mild autonomous cortisol secretion (MACS) demonstrate an increased risk of fractures. As most patients with MACS do not have increased cortisol production, we hypothesized that abnormal circadian cortisol secretion is associated with impaired bone metabolism. Objective: To compare the circadian secretory pattern of bone metabolism biomarkers in relation to cortisol circadian secretion in patients with MACS versus referent subjects. Methods: We conducted a pilot cross-sectional study of patients with MACS and referent subjects. MACS was diagnosed in patients with adrenal adenomas based on an abnormal 1 mg dexamethasone suppression test (cortisol >1.8 mcg/dL). Referent subjects with available abdominal imaging within 5 years and adrenal disorder were recruited from the community. Participants were admitted to the clinical research unit for blood sampling every 2 hours over 24h period. Measurements included total and free cortisol, calcium, parathyroid hormone (PTH), osteocalcin, amino-terminal propeptide type 1 collagen (PINP), and carboxy-terminal telopeptide of type 1 collagen (CTX). Subjects with disorders or medication affecting bone metabolism were excluded. Time periods of blood draw were categorized into the morning (6 to 12 hours), afternoon (14-18 hours) and night (20-2 hours). Delta (morning measurements/afternoon measurements) was calculated. Results: Subjects included 12 patients with MACS (median age 57, IQR 48-67, 83% women) and 10 referent subjects (median age 57, IQR 50-63, 80% women). The 24-hour area under the curve for total (10342 vs 9090 mcg/dL, p=0.468) and free cortisol (437 vs 329 mcg/dL, p=0.086) was no different between patients with MACS and referent subjects. However, the mean night total (6.7 vs 4.3 mcg/dL, p=0.056) and free cortisol (0.2 vs 0.1 mcg/dL, p=0.035) was higher in MACS vs referent subjects. The area under the curve for calcium, phosphorus, CTX, osteocalcin and PINP were similar between patients and referent subjects, however, the area under the curve for PTH was higher in patients vs referent subjects (mean 64179 vs 42602 pg/mL, p=0.021). Both delta total cortisol (mean 8.6 vs 5.5, p=0.01) and delta PTH (mean -1.5 vs 4.2, p=0.051) was higher in referent subjects vs patients with MACS. Conclusion: When compared to referent subjects, patients with MACS have similar 24-hour cortisol production but demonstrate an increase in night-time cortisol secretion and a blunting of diurnal cortisol (lower morning/afternoon cortisol). Patients with MACS have higher PTH production overall, as well as an abnormal diurnal PTH secretion. Noted abnormalities in PTH may contribute to the MACS-associated impairment in bone health. Presentation: Thursday, June 15, 2023

ABSTRACT Opportunistic screening is a new promising technique to identify individuals at high risk for osteoporotic fracture using computed tomography (CT) scans originally acquired for an clinical purpose unrelated to osteoporosis. In these CT scans, a calibration phantom traditionally required to convert measured CT values to bone mineral density (BMD) is missing. As an alternative, phantomless calibration has been developed. This study aimed to review the principles of four existing phantomless calibration methods and to compare their performance against the gold standard of simultaneous calibration (ΔBMD). All methods were applied to a dataset of 350 females scanned with a highly standardized CT protocol (DS1) and to a second dataset of 114 patients (38 female) from clinical routine covering a large range of CT acquisition and reconstruction parameters (DS2). Three of the phantomless calibration methods must be precalibrated with a reference dataset containing a calibration phantom. Sixty scans from DS1 and 57 from DS2 were randomly selected for this precalibration. For each phantomless calibration method first the best combination of internal reference materials (IMs) was selected. These were either air and blood or subcutaneous adipose tissue, blood, and cortical bone. In addition, for phantomless calibration a fifth method based on average calibration parameters derived from the reference dataset was applied. For DS1, ΔBMD results (mean ± standard deviation) for the phantomless calibration methods requiring a precalibration ranged from 0.1 ± 2.7 mg/cm3 to 2.4 ± 3.5 mg/cm3 with similar means but significantly higher standard deviations for DS2. Performance of the phantomless calibration method, which does not require a precalibration was worse (ΔBMD DS1: 12.6 ± 13.2 mg/cm3, DS2: 0.5 ± 8.8 mg/cm3). In conclusion, phantomless BMD calibration performs well if precalibrated with a reference dataset. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

ABSTRACT Estrogen regulates bone mass in women and men, but the underlying cellular mechanisms of estrogen action on bone remain unclear. Although both estrogen receptor (ER)α and ERβ are expressed in bone cells, ERα is the dominant receptor for skeletal estrogen action. Previous studies using either global or cell‐specific ERα deletion provided important insights, but each of these approaches had limitations. Specifically, either high circulating sex steroid levels in global ERα knockout mice or the effects of deletion of ERα during growth and development in constitutive cell‐specific knockout mice have made it difficult to clearly define the role of ERα in specific cell types in the adult skeleton. We recently generated and characterized mice with tamoxifen‐inducible ERα deletion in osteocytes driven by the 8‐kb Dmp1 promoter (ERαΔOcy mice), revealing detrimental effects of osteocyte‐specific ERα deletion on trabecular bone volume (−20.1%) and bone formation rate (−18.9%) in female, but not male, mice. Here, we developed and characterized analogous mice with inducible ERα deletion in osteoclasts using the Cathepsin K promoter (ERαΔOcl mice). In a study design identical to that with the previously described ERαΔOcy mice, adult female, but not male, ERαΔOcl mice showed a borderline (−10.2%, p = 0.084) reduction in trabecular bone volume, no change in osteoclast numbers, but a significant increase in serum CTx levels, consistent with increased osteoclast activity. These findings in ERαΔOcl mice differ from previous studies of constitutive osteoclast‐specific ERα deletion, which led to clear deficits in trabecular bone and increased osteoclast numbers. Collectively, these data indicate that in adult mice, estrogen action in the osteocyte is likely more important than via the osteoclast and that ERα deletion in osteoclasts from conception onward has more dramatic skeletal effects than inducible osteoclastic ERα deletion in adult mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The regulation of bone mineral density (BMD) is highly influenced by genetics and age. Although genome‐wide association studies (GWAS) for BMD have uncovered many genes through their proximity to associated variants (variant nearest‐neighbor [VNN] genes), the cell‐specific mechanisms of each VNN gene remain unclear. This is primarily due to the inability to prioritize these genes by cell type and age‐related expression. Using age‐related transcriptomics, we found that the expression of many VNN genes was upregulated in the bone and marrow from aged mice. Candidate genes from GWAS were investigated using single‐cell RNA‐sequencing (scRNA‐seq) datasets to enrich for cell‐specific expression signatures. VNN candidate genes are highly enriched in osteo‐lineage cells, osteocytes, hypertrophic chondrocytes, and Lepr+ mesenchymal stem cells. These data were used to generate a “blueprint” for Cre‐loxp mouse line selection for functional validation of candidate genes and further investigation of their role in BMD maintenance throughout aging. In VNN‐gene‐enriched cells, Sparc , encoding the extracellular matrix (ECM) protein osteonectin, was robustly expressed. This, along with expression of numerous other ECM genes, indicates that many VNN genes likely have roles in ECM deposition by osteoblasts. Overall, we provide data supporting streamlined translation of GWAS candidate genes to potential novel therapeutic targets for the treatment of osteoporosis. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Abstract The geroscience hypothesis posits that by targeting key hallmarks of aging we may simultaneously prevent or delay several age-related diseases and thereby increase healthspan, or life span spent free of significant disease and disability. Studies are underway to examine several possible pharmacological interventions for this purpose. As part of a National Institute on Aging workshop on the development of function-promoting therapies, scientific content experts provided literature reviews and state-of-the-field assessments for the studies of senolytics, nicotinamide adenine dinucleotide (NAD+) boosters, and metformin. Cellular senescence increases with age, and preclinical studies demonstrate that the use of senolytic drugs improves healthspan in rodents. Human studies using senolytics are in progress. NAD+ and its phosphorylated form, NADP+, play vital roles in metabolism and cellular signaling. Increasing NAD+ by supplementation with precursors including nicotinamide riboside and nicotinamide mononucleotide appears to extend healthspan in model organisms, but human studies are limited and results are mixed. Metformin is a biguanide widely used for glucose lowering, which is believed to have pleiotropic effects targeting several hallmarks of aging. Preclinical studies suggest it improves life span and healthspan, and observational studies suggest benefits for the prevention of several age-related diseases. Clinical trials are underway to examine metformin for healthspan and frailty prevention. Preclinical and emerging clinical studies suggest there is potential to improve healthspan through the use of pharmacologic agents reviewed. However, much further research is needed to demonstrate benefits and general safety for wider use, the appropriate target populations, and longer-term outcomes.

ABSTRACT Most fracture risk assessment tools use clinical risk factors combined with bone mineral density (BMD) to improve assessment of osteoporosis; however, stratifying fracture risk remains challenging. This study developed a fracture risk assessment tool that uses information about volumetric bone density and three‐dimensional structure, obtained using high‐resolution peripheral quantitative compute tomography (HR‐pQCT), to provide an alternative approach for patient‐specific assessment of fracture risk. Using an international prospective cohort of older adults ( n = 6802) we developed a tool to predict osteoporotic fracture risk, called μFRAC. The model was constructed using random survival forests, and input predictors included HR‐pQCT parameters summarizing BMD and microarchitecture alongside clinical risk factors (sex, age, height, weight, and prior adulthood fracture) and femoral neck areal BMD (FN aBMD). The performance of μFRAC was compared to the Fracture Risk Assessment Tool (FRAX) and a reference model built using FN aBMD and clinical covariates. μFRAC was predictive of osteoporotic fracture (c‐index = 0.673, p < 0.001), modestly outperforming FRAX and FN aBMD models (c‐index = 0.617 and 0.636, respectively). Removal of FN aBMD and all clinical risk factors, except age, from μFRAC did not significantly impact its performance when estimating 5‐year and 10‐year fracture risk. The performance of μFRAC improved when only major osteoporotic fractures were considered (c‐index = 0.733, p < 0.001). We developed a personalized fracture risk assessment tool based on HR‐pQCT that may provide an alternative approach to current clinical methods by leveraging direct measures of bone density and structure. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

ABSTRACT Aging is a major risk factor for most chronic diseases, including osteoporosis, and is characterized by an accumulation of senescent cells in various tissues. MicroRNAs (miRNAs) are critical regulators of bone aging and cellular senescence. Here, we report that miR ‐ 19a ‐ 3p decreases with age in bone samples from mice as well as in posterior iliac crest bone biopsies of younger versus older healthy women. miR ‐ 19a ‐ 3p also decreased in mouse bone marrow stromal cells following induction of senescence using etoposide, H 2 O 2 , or serial passaging. To explore the transcriptomic effects of miR ‐ 19a ‐ 3p , we performed RNA sequencing of mouse calvarial osteoblasts transfected with control or miR ‐ 19a ‐ 3p mimics and found that miR ‐ 19a ‐ 3p overexpression significantly altered the expression of various senescence, senescence‐associated secretory phenotype‐related, and proliferation genes. Specifically, miR ‐ 19a ‐ 3p overexpression in nonsenescent osteoblasts significantly suppressed p16 Ink4a and p21 Cip1 gene expression and increased their proliferative capacity. Finally, we established a novel senotherapeutic role for this miRNA by treating miR ‐ 19a ‐ 3p expressing cells with H 2 O 2 to induce senescence. Interestingly, these cells exhibited lower p16 Ink4a and p21 Cip1 expression, increased proliferation‐related gene expression, and reduced SA‐β‐Gal+ cells. Our results thus establish that miR ‐ 19a ‐ 3p is a senescence‐associated miRNA that decreases with age in mouse and human bones and is a potential senotherapeutic target for age‐related bone loss. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.