The cartilaginous growth plate is a critical organ responsible for longitudinal bone growth. It closes after puberty in humans but remains open throughout life in mice. Although cartilage stem cells have been identified in murine growth plates, their existence in humans and their regulation by growth hormone (GH), the most widely used therapy for growth retardation, remain unknown. Here, we characterized the cellular and molecular organization of early pubertal human growth plates using unique surgical specimens from growth-restricting procedures and examined their direct responsiveness to GH. Single-cell and spatial analyses revealed two distinct stemlike populations in the resting zone, differing in proliferative activity, molecular identity, and regulatory cues. The root stem cells express multiple skeletal stem cell markers but not parathyroid hormone-related peptide and reside in a specialized microenvironment low in WNT and TGF-β growth factors. A similar population was identified in transcriptionally profiled unsorted murine growth plates, and clonal lineage tracing demonstrated that these root cells, marked by expression of the Prrx1 gene, generate extensive chondrocyte clones and differentiate into stromal and osteoblastic lineages, confirming their stem cell properties. Human growth plate explant cultures showed that GH directly activates JAK/STAT, TGF-β, and ERK intracellular signaling pathways, inhibits AKT signaling, and stimulates cartilage growth and proliferation of cartilage stem cells and chondrocytes in the proliferative zone. Together, these findings uncover a conserved dual stem cell organization in human and mouse growth plates and define direct mechanisms of GH action, providing a framework for optimizing growth-promoting therapies.

Human bone marrow mesenchymal stromal/stem cells (BM-MSCs) are widely used in clinical trials and tissue engineering, yet their native microenvironment remains poorly understood. Here we introduce a tissue-clearing protocol, DeepBone, for human bones and integrate it with simultaneous mRNA and protein detection. Using this protocol, we spatially map BM-MSCs relative to key bone microenvironment components, including human blood capillaries, adipocytes, sinusoids and bony trabeculae. Quantitative analysis reveals that the native microenvironment of human BM-MSCs in young bone is enriched in vasculature, sinusoids, bone matrix and adipocytes. In contrast, in aged bone, BM-MSCs show no preferential association with bone or adipocytes. Proliferative BM-MSCs are predominantly found along blood vessels. Moreover, we identify a specialized microenvironment for BM-MSCs in young bone, characterized by sinusoids coiled around trabeculae and enriched by R-type vessels. These findings provide insights into the native niches of BM-MSCs, offering a foundation for the development of tissue engineering strategies that mimic their physiological context.

Despite the importance of the gut microbiome to health, the role of human genetic variation in shaping its composition remains poorly understood. Here we report genome-wide association analyses of harmonized metagenomic data from 16,017 adults in four Swedish population-based studies, with replication in 12,652 people from the Norwegian HUNT study. We identified variants in the OR51E1-OR51E2 locus, encoding sensors for microbiome-derived fatty acids, associated with microbial richness. We further identified 15 study-wide significant genetic associations (P < 5.4 × 10-11) involving eight loci and 14 common bacterial species, of which 11 associations at six loci were replicated. The results confirm previously reported associations at LCT, ABO and FUT2, and provide evidence for new loci MUC12, CORO7-HMOX2, SLC5A11, FOXP1 and FUT3-FUT6, with supporting data from metabolomics and gene expression analyses. Our findings link gut microbial variation genetically to gastrointestinal functions, including enteroendocrine fatty acid sensing, bile composition and mucosal layer composition.

Abstract Weight‐loading reduces body fat, but its effects on cardiac autonomic modulation remain unclear. We examined heart rate variability (HRV) during free‐living weight‐loading in adults with class I obesity. Fifty‐one participants (27 females) were randomized to wear a heavy (11% of body weight) or a light vest (1%) for 8 h/day for 15 days. Twenty‐four‐hour ECG recordings were obtained before and on day 15. Heart rate (HR), relative HR reserve (%HRR), and time‐ and frequency‐domain HRV indices were analyzed across four predefined periods: morning (rest), afternoon (vest‐usage), evening, and night. During the afternoon period, the high versus low load increased HR (p = 0.0496) and %HRR (p = 0.03), while reducing SDNN (standard deviation of normal‐to‐normal intervals; p = 0.01) and RMSSD (root mean square of successive differences; p = 0.002). In females, but not in males, high load was associated with lower high‐frequency power (PHF), higher low‐frequency power (PLF), and an increased PLF/PHF ratio during the afternoon, consistent with reduced parasympathetic modulation. No differences between groups were observed during resting periods. These findings indicate that weight‐loading increases cardiovascular workload and transiently alters autonomic modulation during active vest use, with more consistent responses in females and no sustained resting adaptations.

Aims The present study aimed to evaluate the association between body mass index (BMI) during childhood and puberty and blood pressure and hypertension in midlife and to explore midlife BMI as a potential mediator of these associations. Methods and results We linked the BMI Epidemiology Study Gothenburg with developmental BMI, with the Swedish CArdioPulmonary bioImage Study (SCAPIS) with blood pressure and hypertension in midlife (n = 2394). The associations between childhood BMI (7-8 years) and pubertal BMI change (young adult BMI minus childhood BMI), and blood pressure and hypertension in midlife, were evaluated using linear or logistic regression models. Mediation analysis was conducted to evaluate the indirect effect, via midlife BMI, and the direct effect on blood pressure and hypertension. The analyses were adjusted for birth year and smoking. The pubertal BMI change was positively associated with systolic and diastolic blood pressures and hypertension in midlife, independent of childhood BMI, in both men and women (P < 0.01). For men but not for women, childhood BMI was positively associated with systolic and diastolic blood pressures in midlife, independent of the pubertal BMI change (P < 0.01). No significant independent association was observed for childhood BMI with hypertension. Mediation analyses for the association between the pubertal BMI change and blood pressure and hypertension in midlife indicate that these associations were largely mediated by BMI in midlife. Conclusion These findings indicate that high blood pressure may originate in early life. A life-course approach for targeted prevention, starting already during the developmental years, could reduce the risk of high blood pressure.

Abstract Background and Aims Pregnancy requires dynamic, stage-specific adaptations in maternal liver metabolism and growth to sustain fetal development while preserving systemic homeostasis. Estrogen signaling, which significantly increases during pregnancy, is primarily mediated in hepatocytes by estrogen receptor α (ERα). Although hepatic ERα regulates female liver metabolism under non-pregnant conditions, its role in pregnancy-induced hepatic remodeling remains unclear. Methods We studied non-pregnant and pregnant control and liver-specific ERα knockout (LERKO) mice across gestational stages using longitudinal physiological measurements, liver transcriptomics, targeted metabolomics, histological assessment of cell proliferation, and metabolic phenotyping. Results In control mice, pregnancy elicited sequential hepatic remodeling characterized by early induction of cell-cycle programs, a mid-gestational peak in hepatocyte proliferation with transient suppression of selected metabolic pathways, and late reactivation of specific metabolic programs. Chronic hepatic ERα deficiency alters this temporal pattern. LERKO livers showed premature activation of proliferative and anabolic transcriptional programs, changes in amino acid- and fatty acid-related metabolic pathways, and altered temporal regulation of AKT-mTORC1-related signaling. At mid-gestation, LERKO mice displayed reduced hepatocyte proliferation, altered expression of metabolic and insulin-related genes, blunted gestational glucose adaptation without overt evidence of systemic insulin resistance, and changes in the light/dark-phase metabolic patterns. Conclusions These findings suggest that hepatic ERα is required for the appropriate stage-specific coupling of liver growth, metabolic remodeling, and insulin-responsive signaling during pregnancy. Its loss is associated with gestational hepatic maladaptation and systemic metabolic phenotypes, providing a framework for investigating estrogen-dependent mechanisms underlying pregnancy-associated metabolic and liver disorders. Highlights Hepatic ERα is required for stage-specific liver remodeling during pregnancy. Loss of hepatic ERα alters temporal coupling of liver growth and metabolism. LERKO mice show early changes in amino acid- and fatty acid-related pathways. Hepatic ERα loss reduces proliferation and alters gestational glucose adaptation. Hepatic ERα loss is associated with altered light/dark-phase metabolic organization. Graphical abstract

Polycystic ovary syndrome (PCOS) is associated with skeletal muscle insulin resistance, fibrosis, and lipotoxicity, yet the cellular origins remain unknown. Here, we present a comprehensive cellular atlas of skeletal muscle from hyperinsulinemic and hyperandrogenic women with PCOS and controls of similar age, weight, and BMI. Analysis of 72,247 nuclei from 19 biopsies revealed cell-type-specific dysregulation in PCOS, with fiber-type-specific metabolic impairment, converging with pro-fibrotic reprogramming of fibro-adipogenic progenitors (FAPs) and enhanced FAP-myofiber crosstalk, characterized by enhanced collagen and laminin signaling. Metformin intervention for 16-weeks selectively reversed PCOS-associated transcriptional dysregulation in FAPs, revealing heterogeneous cellular responses in skeletal muscle. In vitro, PCOS myotubes retained metabolic dysfunction, yet show normalized glucose responsiveness, indicating plasticity despite metabolic memory. Systemic hyperinsulinemia and hyperandrogenemia correlated with transcriptional signatures in muscle fibers and FAPs, linking endocrine imbalance to pro-fibrotic remodeling. These findings identify novel therapeutic targets beyond conventional insulin-sensitizing approaches.

Obesity is a complex disease driven by multiple factors, and a deeper understanding of its underlying mechanisms could enable the development of novel treatments. Based on our previous experimental studies, we have proposed a homeostatic mechanism regulating adiposity involving mechano-sensing of body weight by osteoblast-lineage cells in the lower extremities. However, the molecular mechanism underlying this proposed weight-sensing pathway remains to be elucidated. Recent studies have demonstrated that Piezo1-mediated mechano-sensing in osteoblast-lineage cells, as well as TrkA-dependent signaling, are essential for the normal bone anabolic response to high-intensity mechanical loading. We hypothesized that these pathways within bone may also contribute to the sensing of sustained increased weight loading, thereby influencing the homeostatic regulation of body weight. To test this hypothesis, we first established a high-fat diet-induced obesity mouse model with conditional deletion of Piezo1 in osteoblast-lineage cells. Our results demonstrate that the effect of increased weight loading, induced by implanted weights, on body weight reduction is partially dependent on Piezo1 expression in osteoblast lineage cells. Similarly, using a mouse model lacking functional TrkA signaling, we demonstrated that the response to increased weight loading on body weight reduction is partially dependent on functional TrkA signaling. In conclusion, we demonstrate that the effect of increased weight loading on body weight is at least partially dependent on Piezo1 expression in osteoblast-lineage cells and intact TrkA signaling. Based on these findings we propose that increased body weight, resulting from adiposity, may be sensed by osteoblast-lineage cells through Piezo1 activation and that intact TrkA function is necessary for the weight-reducing response to increased weight loading. This mechanosensory input may then initiate compensatory central pathways that reduce adiposity and body weight.

Osteoporosis is characterized by an imbalance in bone remodeling, resulting in bone loss and increased fracture risk. Inflammatory diseases, such as rheumatoid arthritis, are strongly associated with secondary osteoporosis due to inflammation-induced bone loss. Pro-inflammatory cytokines, particularly TNF-α, disrupt bone homeostasis by promoting osteoclastogenesis and inhibiting osteoblast function. The Wnt signaling pathway is essential for bone formation and is suppressed in inflammatory conditions. WNT16, an osteoblast-derived ligand, increases bone mass mainly by inhibiting osteoclast differentiation but has also been found to stimulate osteoblast activity. Here we demonstrate that TNF-α downregulates Wnt16 mRNA expression in primary osteoblasts, suggesting that inflammation may impair WNT16 expression and thereby reduce bone mass. To evaluate whether pharmacological or genetical elevation of WNT16 levels can mitigate inflammation-induced bone loss, we examined the effect of WNT16 in three mouse models of local and systemic inflammation. In a knee arthritis model, intra-articular delivery of WNT16 liposomes failed to prevent local bone loss. Similarly, although osteoblast-specific WNT16 overexpression increased the overall bone mass, it did not protect against either local calvarial bone loss or systemic bone loss induced by Toll-like receptor 2 (TLR2) activation. Furthermore, in a model of systemic inflammation induced by Staphylococcus aureus, WNT16 overexpression did not preserve vertebral trabecular bone, despite increased baseline bone mass. These findings demonstrate that WNT16, although increasing the overall bone mass, is insufficient to counteract inflammation-driven bone loss.

Complement component 9 (C9) is the main pore-forming protein of the terminal membrane attack complex (MAC) in the complement system. The MAC forms a lytic pore in the membrane of target cells and is a key effector of the innate immune system. Excessive complement activation has been implicated in chronic inflammation, which is a well-established risk factor for increased fracture susceptibility. Although other components of the complement system have been linked to bone mass regulation, the role of C9 is unknown. The aim of the present study was to determine the role of C9 for bone health. To determine the role of circulating, mainly liver-derived, C9 in humans, we conducted Mendelian randomization (MR) using a single-SNP instrument and performed genetic colocalization analyses. Genetically predicted higher circulating C9 levels were causally associated with increased risk of forearm fractures and fractures at any bone site. Colocalization analyses supported these findings. Mechanistic studies were performed in mice. As expected, high C9 expression was observed in the liver, while the expression was low in cortical bone and no expression was observed in trabecular bone. Adult female mice with global C9 deletion (C9-/-) exhibited lower vertebral trabecular bone volume fraction due to a decreased trabecular number, but they were partly protected against ovariectomy-induced trabecular bone loss. No skeletal phenotype was observed in the femur or in male C9-/- mice. In conclusion, high genetically determined circulating C9 is causally linked to increased fracture risk in humans. In mice, global inactivation of C9-/- affects vertebral trabecular bone in a sex-specific and context-dependent manner.

As populations age worldwide, the incidence of fractures is increasing, leading to considerable disease burden and increased health-care costs. Bone mineral density (BMD), bone quality and muscle function are major contributors to fracture risk, with muscle function having a critical role in preventing falls. The gut microbiota is well established as a regulator of these parameters in rodents, with the immune system and short-chain fatty acids acting as key mediators of these effects. First-generation probiotic interventions have demonstrated modest effects on BMD in clinical trials. In humans, the association between the gut microbiota and muscle mass, muscle function and fracture risk seems to be more robust than the correlation between gut microbiota and BMD. Future research should prioritize the development of more targeted gut microbiota-based interventions, including second-generation probiotics, personalized dietary strategies, synbiotics, postbiotics and species-specific antibiotics with the potential to enhance both BMD and muscle function, thereby reducing risk of fractures.

Polycystic ovary syndrome (PCOS) is characterized by reproductive and metabolic disturbances and associated with higher prevalence of anxiety and depression. Circulating adiponectin, an insulin-sensitizing adipokine, is reduced in women with PCOS, and low adiponectin has been linked to impaired mental health, particularly in females. We investigated whether low serum adiponectin is associated with impaired mental health in women with PCOS and whether adiponectin deficiency exacerbates anxiety-like behaviour in a PCOS-like mouse model. Serum adiponectin was measured in women with (n = 179) and without PCOS (n = 228), stratified by body mass index (BMI). Health-related quality of life was assessed using the short form 36 (SF-36), generating physical and mental component scores. In parallel, the prenatal androgenization (PNA) PCOS-like mouse model was combined with adiponectin-deficient mice (APNhet) to assess the impact of reduced adiponectin on anxiety-like behaviour with and without prenatal androgen exposure. Women with PCOS had lower total and high molecular weight adiponectin levels compared with controls. Adiponectin positively correlated with mental component scores in women with BMI < 30, but not in those with obesity. Free testosterone was inversely correlated with adiponectin. In mice, PNA induced anxiety-like behaviour. However, despite a 65% reduction in serum adiponectin levels in APNhet PNA mice, adiponectin deficiency did not exacerbate the anxiety-like phenotype. Unlike women with PCOS, adult PNA mice were not hyperandrogenic. These findings suggest that adiponectin is associated with mental health in non-obese women, but reduced adiponectin alone does not induce anxiety-like behaviour in the absence of hyperandrogenism. Further studies in hyperandrogenic models are needed to clarify how adiponectin and hyperandrogenism interact across developmental stages and in different BMI groups to influence vulnerability to anxiety and depression in PCOS.Trial registrationClinical trial.gov: NCT01319162, NCT00484705, NCT01457209, and NCT00921492.

Importance:Childhood overweight is associated with adult coronary heart disease (CHD); the extent to which this risk can be mitigated by remission of childhood overweight before young adulthood is not clear. Objective:To evaluate if remission of elevated childhood weight before young adulthood mitigates the risk of adult CHD. Design, Setting, and Participants:This population-based cohort study was conducted among individuals born between 1945 and 1968 in Gothenburg, Sweden, as a part of the BMI Epidemiology Study (BEST). Archived child and school health records were linked to national high-quality registers in November 2022. Data analysis was performed from 2024 to 2025. Exposures:Childhood (women at age 7 years and men at age 8 years) and young adult (women age 18 years and men age 20 years) overweight derived from weight and height measurements in school health records and at conscription. Main Outcomes and Measures:The primary outcome was register-derived CHD diagnosis (fatal or nonfatal) in adult age. Results:This study included 103 232 individuals (45 965 women [44.5%]; mean [SD] childhood body mass index, calculated as weight in kilograms divided by height in meters squared, of 15.6 [1.5]) born 1945-1968 in Gothenburg, Sweden. Childhood and young adult overweight were associated with increased risk of CHD in both men and women. No significant sex interaction was observed for these associations. Remission of childhood overweight before young adulthood resulted in a similar risk of CHD as in individuals who had persistent normal weight (reference category; hazard ratio [HR], 0.98; 95% CI, 0.84-1.14). Both pubertal onset overweight (ie, normal weight in childhood and overweight in young adulthood; HR, 1.83; 95% CI, 1.66-2.03) and persistent overweight (ie, overweight in both childhood and young adulthood; HR, 1.53; 95% CI, 1.30-1.78) were associated with increased risk of adult CHD events. However, individuals with pubertal onset overweight had higher risk of CHD than individuals with persistent overweight (HR, 1.23; 95% CI, 1.03-1.49; P = .03). Conclusions and Relevance:In this population-based cohort study, increased risk of CHD in Swedish individuals with childhood overweight was reversed with remission of overweight before young adulthood; furthermore, overweight in young adulthood with pubertal onset was associated with higher risk of adult CHD compared to overweight persistent throughout childhood and puberty. These findings have implications for public health planning, emphasizing the importance of early detection and treatment of overweight during childhood and adolescence.

Hip fractures are a significant public health concern, associated with high morbidity and mortality. This population-based cohort study investigated whether COVID-19 increased hip fracture risk in Swedish adults. The study included 3,931,893 Swedish residents aged ≥50 years as of January 1st 2020, and recorded 50,883 incident hip fractures during follow-up, until the end of 2022. The exposure of interest was COVID-19 confirmed via national registries during the study period, with 711,879 (18.1%) identified cases. The primary outcome was hip fracture incidence. Secondary outcomes included fractures of the proximal humerus and wrist. Association between COVID-19 and fractures was estimated using Cox proportional hazards regression with time-varying exposure, adjusting for demographic and socioeconomic characteristics, frailty index, previous comorbidities, prior medication use, and COVID-19 vaccination status. Results were presented as hazard ratios (HRs) with 95% confidence intervals (CIs). In fully adjusted models, COVID-19 was associated with a 43% higher risk of hip fractures (HR 1.43, 95% CI 1.36-1.50), with stronger association observed in men (HR 1.57, 95% CI 1.45-1.71) than in women (HR 1.36, 95% CI 1.28-1.45). Age-stratified analyses revealed significant associations in individuals aged ≥65 years (men: HR 1.68, 95% CI 1.54-1.82; women: HR 1.42, 95% CI 1.33-1.51), but not in those aged 50-65 years. Associations with proximal humerus and wrist fractures were less pronounced. These findings suggest that COVID-19 is associated with a substantially increased risk of hip fracture, especially among individuals aged ≥65 years old, underscoring the importance of targeted fracture prevention in this group after COVID-19.

Kidney size is sex-dimorphic and regulated by androgens in adult humans and mice. However, the effects of developmental androgen deficiency on kidneys remain elusive. We hypothesized that androgens program future kidney growth during fetal development. Male mice lacking the main testosterone-producing enzyme HSD17B3 had reduced testosterone at embryonic day 15.5, but the concentrations increased by E18.5, creating a short time window of androgen deficiency resulting in reduced kidney size in adult males. In male Hsd17b3 -/- kidneys, nephron development was qualitatively normal, but the number of glomeruli and proliferation of proximal tubules were reduced, as was proximal tubule size in adults. Testosterone supplementation at E14.5-17.5 normalized the renal size in adult Hsd17b3 -/- males. Our data suggest that androgen receptor and HNF4A jointly regulate IGFBP5, putatively influencing FOXO1 and mTOR signaling to promote male-specific kidney growth in the fetal period. In conclusion, we have identified a novel developmental programming effect on male kidneys, where fetal androgen deficiency reduces kidney growth and androgen responsiveness in adult males.

Aging is associated with reduced lean and bone mass, as well as alterations in gut microbiota composition. We previously demonstrated that gut microbiota composition differs between young adult and old mice, and that transplanting gut microbiota from old donors into young germ-free mice reduces lean mass, but not bone mass, compared with transplantation from young adult donors. In this study, we investigated whether the reduced lean and bone mass observed in old mice could be restored through gut microbiota transplantation from young adult donors. Old mice (18-mo old) were treated with antibiotics to deplete their gut microbiota and subsequently transplanted with gut microbiota from either young adult (5-mo old) or old (21-mo old) donors. Recipient mice colonized with gut microbiota from young adult donors showed distinct beta and alpha diversity compared with those colonized with gut microbiota from old donors, demonstrating successful transplantation. However, no differences in lean or bone mass were observed between old mice transplanted with gut microbiota from young adult donors and those receiving gut microbiota from old donors. In conclusion, our findings demonstrate that gut microbiota composition differs in mice transplanted with young adult compared with old gut microbiota but neither reduced lean mass nor reduced bone mass in old mice can be restored through gut microbiota transplantation from young adult donors.NEW & NOTEWORTHY Aging is associated with reduced lean and bone mass and changes in gut microbiota (GM). We tested whether transplanting young adult GM could reverse these age-related conditions in old mice. GM transplantation resulted in distinct GM compositions between mice receiving young adult versus old donor GM, but neither lean nor bone mass was restored in old mice. These findings suggest that GM from young adult mice cannot restore musculoskeletal deficits in aging.

In this meta-analysis of international cohorts, current smoking is confirmed as a significant BMD-independent predictor of future fracture with a stronger relationship in men than in women. A causative and reversible effect of smoking on fracture risk is suggested by past smoking having a significantly lower risk than current smoking. In this meta-analysis of international cohorts, the aim was to examine the relationship of current and past smoking with fracture risk to provide an update for future iterations of the FRAX tool. The risk of fracture associated with current and past smoking was estimated using an extended Poisson model applied separately to each of 58 prospective international cohort studies. Covariates included current time since start of follow up, current age, and in an additional model, BMD at the femoral neck. The results of the different studies were merged by using inverse-variance weighted β-coefficients. This analysis included a total of 1,691,024 participants (61.2

Epidemiological data have indicated an increased risk of inflammatory bowel disease associated with the female sex and contraceptive hormones. However, in preclinical models of estradiol in colitis, results have been inconsistent, and both alleviating and aggravating effects have been reported. Previously, we suggested that the estrogen receptor α mediates inflammation in the colon. Here, we investigated the effects of estradiol and progesterone in dextran sulphate sodium-induced colitis, using sham-operated or ovariectomized female mice with or without hormonal supplementation initiated three weeks before inflammation. We found that sham-operated, or ovariectomized mice supplemented either with only estradiol or estradiol and progesterone exhibited more severe intestinal inflammation compared to ovariectomized mice at day 7 of colitis. Simultaneously, progesterone supplementation of ovariectomized mice did not affect the inflammatory status. Estrogen receptors were expressed in several different cell types of the colonic mucosa, although at low levels. The estrogen receptor α was observed in the epithelium as well as in mononuclear phagocytes and T and B cells. The estrogen receptor β was found in the epithelium, and the G protein-coupled estrogen receptor in the lamina propria and vessel structures. Further, hormone depletion increased homeostatic immune cells, while estradiol exposure altered the expression of 791 genes involved in pathways promoting the antimicrobial response, immune cell activation, and metabolism related to epithelial integrity. With our results, we are the first to present strong and detailed evidence of estradiol as an inflammatory agent in the colon of female mice when allowing the appearance of longer-term effects.