BACKGROUND:Metabolic reprogramming is increasingly recognized as a key driver of endocrine tumor biology, yet how dysregulated metabolism promotes aldosterone excess in aldosterone-producing adenomas remains largely unclear. METHODS:Multiomics profiling of human adrenal and blood specimens was performed to identify metabolic reprogramming. Dysregulated genes and accumulation of intermediates in the propionate metabolism were validated in aldosterone-producing adenomas. Functional effects on aldosterone production were assessed in adrenocortical cells and mice. Key downstream molecules and pathways were examined through transcriptomics, mass spectrometry, and targeted functional assays. RESULTS:Aldosterone-producing adenomas exhibited reprogrammed propionate metabolism and accumulation of its byproduct methylmalonic acid, associated with upregulation of the upstream enzyme PCCA (propionyl-CoA carboxylase subunit A). In adrenocortical cells, PCCA overexpression increased CYP11B2 (aldosterone synthase) expression and aldosterone production, while its silencing had the opposite effects. In both adrenocortical cells and female mouse adrenals, methylmalonic acid promoted CYP11B2 expression and aldosterone production. Mechanistically, methylmalonic acid elevated reactive oxygen species, which triggered S-glutathionylation modification of the L-type calcium channel Cav1.2 at cysteines 106 and 621, as confirmed by mass spectrometry and site-directed mutagenesis. This redox-dependent modification enhanced Cav1.2 plasma membrane expression and intracellular calcium concentrations, thereby activating calcium/calmodulin signaling, upregulating CYP11B2 expression, and driving aldosterone production. CONCLUSIONS:These findings identify a methylmalonic acid-oxidative stress-Cav1.2 S-glutathionylation axis that links metabolic rewiring to aldosterone production, uncovering potential therapeutic opportunities targeting redox-dependent metabolic signaling in primary aldosteronism.

A proportion of patients with new-onset diabetes share similar symptoms with type 1 diabetes (T1D) patients but they are negative for islet antigen-specific autoantibodies. This study was to develop an islet antigen-specific CD8+ T-cell assay to provide autoimmune evidence regarding these “suspected” T1D patients. HLA-A*0201 individuals with autoAbs+ T1D, autoAbs− suspected T1D, and type 2 diabetes, along with HLA-A*0201 healthy controls were recruited. Using interferon-γ enzyme-linked immunospot assays, the percentages of participants in each group with various islet antigen-specific CD8+ T cells were determined. Sixteen out of the 28 islet antigen-specific epitopes tested were T1D specific, meaning that there was a significantly (P < 0.05) greater epitope positivity rate in the autoAbs+ T1D cohort than in the healthy controls. Using a cutoff value of two positive epitopes, the 16-epitope panel led to a sensitivity of 75.0

Background:Heart failure (HF) is a severe and common complication of type 2 diabetes mellitus (T2DM), associated with increased morbidity and mortality. Although the biomarker NT-proBNP, at a cut-off value of 125 pg/mL, has demonstrated satisfactory discriminatory power for predicting HF risk in T2DM patients, its measurement remains inaccessible in most primary healthcare settings in China. This study aimed to develop and externally validate a machine learning-based nomogram for predicting the risk of elevated NT-proBNP (≥125 pg/mL) as a surrogate for HF risk in patients with T2DM. Methods:We retrospectively enrolled 564 T2DM patients as the development cohort and 302 from two external centers as the validation cohort. After feature selection via least absolute shrinkage and selection operator regression, five machine learning models were constructed and evaluated using 10-fold cross-validation. The optimal model was presented as a static nomogram and further deployed as an online web application for clinical use. Results:Six key predictors were identified: estimated glomerular filtration rate, age, serum albumin, hemoglobin, urine albumin-to-creatinine ratio, and the binary indicator of age ≥ 65 years. Interpretability analysis using SHapley Additive exPlanations revealed estimated glomerular filtration rate as the most influential feature. The final machine learning-based nomogram achieved AUCs of 0.806 (95% CI: 0.767-0.845) in training and 0.861 (95% CI: 0.813-0.908) in external validation, with good calibration and clinical utility. Furthermore, the nomogram scores showed a significant positive correlation with established TRS-HFDM risk strata, supporting its clinical relevance. Conclusion:We developed and validated an interpretable machine learning-based nomogram that effectively predicts the risk of elevated NT-proBNP in T2DM patients using six routine clinical variables. This tool demonstrates robust performance and generalizability, offering a practical and accessible solution for HF risk stratification in resource-limited primary care settings in China.

Disclosure: Y. Yang: None. Y. Yan: None. T.A. Williams: None. G. Ma: None. M. Gao: None. Y. Liu: None. Z. Zhang: None. M. Sun: None. Background: The investigation of resected adrenals from patients with primary aldosteronism has revealed various aldosterone-producing lesions with distinct genotypes. These lesions may represent different stages of adenoma development. We employed genetic and spatial transcriptomic analyses to determine how transcriptomic alterations, potentially driven by aldosterone-driver mutations, may contribute to adenoma formation. Methods: Formalin-fixed paraffin-embedded adrenal sections from 53 patients with unilateral primary aldosteronism were consecutively analyzed. Aldosterone synthase immunohistochemistry-guided DNA sequencing was performed. Spatial transcriptomic analyses were conducted on 12 aldosterone-producing lesions with distinct genotypes, and integrated with public single-cell and single-nucleus transcriptomes. Results: Somatic mutations were identified in 96.6% (28/29) of aldosterone-producing adenomas (APAs) and 87.5% (14/16) of aldosterone-producing nodules, with KCNJ5 mutations being the most frequent (89.7% vs 50.0%, respectively). In aldosterone-producing micronodules (APMs), similar numbers of KCNJ5 (n=15) and CACNA1D mutations (n=12) were identified. Spatial transcriptomics revealed intratumoral transcriptional heterogeneity in APAs with KCNJ5 mutations (n=2) and identified a unique cluster of cell populations with high SHH expression. APMs with KCNJ5 (n=3), CACNA1D mutations (n=4) or no mutation detected (n=3) exhibited similar activated pathways and cell compositions. The SHH pathway was enriched exclusively at the boundaries of APMs with KCNJ5 mutations, suggesting that morphogenic signals from adjacent cells support APM-to-APA transition in the presence of the same mutation. Conclusions: Somatic KCNJ5 mutations are not rare in APMs in Chinese patients with unilateral primary aldosteronism. APAs with KCNJ5 mutations share signaling pathways with cells adjacent to APMs with KCNJ5 mutations, highlighting genotype-dependent capacities for adenoma formation. Presentation: Saturday, July 12, 2025

Purpose: Pituitary adenoma and its treatments can significantly impact the social function of young and middleaged patients. Monitoring social functioning trajectories over time provides a more holistic understanding of patients' quality of life. We aimed to study the social functional trajectories and their predictors to achieve individualized and comprehensive management. Methods: A longitudinal prospective survey was conducted from January 2021 to December 2022 in Nanjing, China. The convenience sampling method was adopted to recruit the patients with pituitary adenoma who underwent transsphenoidal surgery at the neurosurgery department in a tertiary-grade A hospital. The general information questionnaire, social disability screening scale and postoperative pituitary adenoma symptom distress scale were applied in different stages. Growth mixture modeling and the Logistic regression were utilized to identify the trajectory subgroups and their predictors of the social function. Results: Of 225 patients, 33 (14.67%) experienced "progressively recovered function", while 192 (85.33%) exhibited "persistently impaired social function". Factors associated with persistently impaired social function included fertility status (OR = 0.167, p = 0.001), visual loss (OR = 0.154, p = 0.012), and electrolyte disturbances at 6th month (OR = 0.047, p = 0.003). Conversely, patients with high monthly income (OR = 0.116, p = 0.000), good family support (OR = 3.890, p = 0.006), and preoperative headache (OR = 4.129, p = 0.006) were more likely to show progressively recovered function after surgery. Conclusions: Patients with pituitary adenoma still experience social dysfunction six months after surgery. Fertility status, family support, economic status, and symptom distress were significant predictors. This study emphasizes the crucial role of symptom distress and family support, providing a basis for follow-up and intervention strategies.

Disclosure: Y. Yang: None. M. Gao: None. Z. Xia: None. Y. Liu: None. Z. Zhang: None. M. Sun: None. Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism, characterized by upregulation of CYP11B2 gene transcription (encoding aldosterone synthase) and aldosterone biosynthesis in adrenal zona glomerulosa cells. While alterations in metabolic pathways have been reported in APAs, their roles in aldosterone production remain largely undefined. Here, we identified dysregulated propionate metabolism in APAs compared with adjacent zona glomerulosa by bioinformatic analyses using a publicly available dataset (GSE64957). The levels of methylmalonic acid (MMA), a by-product of propionate metabolism, were upregulated in the venous serum from the ipsilateral side of adrenals compared with the contralateral side in patients with APAs (n=15 pairs). This occurred possibly through the upregulation of propionyl-CoA carboxylase alpha (PCCA) in APAs confirmed by immunoblotting and immunohistochemistry. In human adrenocortical cells (HAC15), overexpression or silencing of PCCA caused upregulation or downregulation of CYP11B2 expression and aldosterone production, respectively. MMA treatment in HAC15 cells or C57BL/6 mice also increased CYP11B2 expression and aldosterone production. RNA sequencing revealed an MMA-induced alteration of calcium signaling in HAC15 cells. The elevation of intracellular calcium concentration was later confirmed in MMA-treated cells, and the stimulatory effect of MMA on CYP11B2 expression was reversible by calcium inhibitor nifedipine and calmodulin kinase II inhibitor KN93. Coherently with previous findings, we identified an elevated reactive oxygen species level following MMA treatment. Given the relationship between reactive oxygen species generation and glutathionylation, and the enrichment of the Gene Ontology term “Glutathione binding” in RNA sequencing, we demonstrated that MMA induced glutathionylation in HAC15 cells. Notably, the glutathionylation of L-type calcium channel Cav1.2, which was reported to regulate intracellular calcium influx, was enhanced by MMA. This effect was reversible by the reactive oxygen species scavenger N-acetylcysteine. Mass spectrometry further confirmed that MMA induced the glutathionylation of Cav1.2 at positions Cysteine 106 and Cysteine 621. Collectively, we present a previously uncharacterized dysregulation of propionate metabolism as an important contributor to aldosterone production via MMA-induced Cav1.2 glutathionylation. This pathway represents a potentially valuable target for the development of new therapeutic strategies for primary aldosteronism. Presentation: Sunday, July 13, 2025

Primary aldosteronism (PA) caused by aldosterone hypersecretion is treated by adrenalectomy or medications. Histopathologic examination of resected adrenals reveals diverse histopathologic features. This study aimed to investigate the potential association of peripheral and adrenal tissue metabolic profiles with the histopathologic features of PA. The retrospective study included 105 surgically treated and 43 medically treated patients with PA. Adrenal specimens were categorized according to the HISTALDO (HISTopathology of primary ALDOsteronism) consensus. Peripheral and adrenal tissue metabolic profiles were assessed, including adiposity, adipokines and fatty acid abundances. The distinct fatty acid, arachidonic acid, was further functionally characterized. Surgically treated patients with classical histopathologic findings (n = 71) displayed lower body mass indexes, a lower prevalence of obesity, smaller waist circumference and visceral adipose tissue areas, and lower leptin concentrations compared with operated patients with the nonclassical histopathology (n = 34). No such differences were identified between the nonclassical histopathology group and medically treated group. Distinct concentrations of 18 out of 35 peripheral venous fatty acids, including arachidonic acid, were identified among the 3 groups. Further, accumulation of arachidonic acid was demonstrated in 4 aldosterone-producing adenomas compared with paired adjacent cortex possibly linked with suppressed peroxisomal beta-oxidation. Stimulation of human adrenocortical cells with arachidonic acid or peroxisomal beta-oxidation inhibitor caused 3.8-fold (P = 0.0050) and 1.7-fold (P = 0.0328) amplification of CYP11B2 expression, respectively, which were ablated by BAPTA-AM or KN93, and induced oxidative stress and apoptosis. Our findings show metabolic heterogeneity related to histopathology and support a role for arachidonic acid in PA pathophysiology.

ObjectiveTo analyze the clinical characteristics of immune checkpoint inhibitor (ICI)-related hypophysitis. MethodsA retrospective analysis was conducted on patients diagnosed with ICI-related hypophysitis and treated at the Department of Endocrinology, The First Affiliated Hospital with Nanjing Medical University, between January 2020 and March 2025. Clinical manifestations and prognosis of patients were analyzed. ResultsEleven patients with ICI-related hypophysitis were included. The average age was (62.27±7.63) years, and 9 patients (81.82%) were male. The median time to onset was 9.1 months, and the median number of treatment cycles received was 5. The primary initial symptoms were fatigue and anorexia. Hyponatremia was present in 3 patients (27.27%). Evaluation of anterior pituitary function revealed adrenocorticotropic hormone deficiency as the most common manifestation (90.91%, 10/11), followed by hyperprolactinemia (81.82%, 9/11). Posterior pituitary function remained normal in all patients. Pituitary magnetic resonance imaging showed no abnormality in 4 patients (44.44%). Thyroid dysfunction was observed in 6 patients (54.55%), one of whom (9.09%) also exhibited pancreatic endocrine dysfunction. The average follow-up duration was 36.5 months. Eight patients (72.73%) were alive at the last follow-up. None of the patients recovered their pituitary hormone function. ConclusionsEndocrine adverse events induced by ICIs can involve multiple glandular systems. Clinicians should be highly vigilant for the possibility of ICI-induced hypophysitis in patients receiving ICIs who present with symptoms such as fatigue, anorexia, and hyponatremia.

BackgroundRenin-independent aldosterone secretion contributes to aldosteronism and heightened cardiovascular risk, but renin-independent aldosteronism is highly heterogenous. A refined classification may assist in identifying individuals with distinct cardiovascular risk profiles and guide individualized treatment strategies.MethodsUnsupervised hierarchical clustering was performed using 12 clinical parameters from patients with renin-independent aldosteronism in our registry cohort (n=404). The cluster centroids derived from the discovery cohort were fixed and applied to the Framingham Heart Study Third Generation cohort (n=417) for subject classification. The identified clusters were evaluated for their association with cardiovascular outcomes, assessed by echocardiographic parameters, serum biomarkers and cardiovascular event rates.ResultsThree replicable clusters of patients with renin-independent aldosteronism were identified. Patients in cluster 2 showed the most severe metabolic abnormalities with the highest lipid and glucose levels, while patients in cluster 3 displayed the highest aldosterone levels. Both clusters 2 and 3 showed elevated baseline blood pressure and left ventricular remodeling compared with cluster 1. Cluster 2 exhibited the highest risk of cardiovascular disease, chronic heart failure and atrial fibrillation, followed by cluster 3, which showed a higher incidence of cardiovascular disease compared with cluster 1.ConclusionsWe identified 3 subgroups with differing degrees of target organ damage and cardiovascular risk. Our findings establish metabolic dysfunction, rather than aldosterone excess, as a potential dominant cardiovascular risk driver in RIA patients, defining a new risk paradigm. Patients with renin-independent aldosteronism with metabolic dysfunction or high aldosterone levels may benefit from mineralocorticoid receptor antagonists with different priorities for metabolic and cardiovascular protection. This new refined classification may help tailor optimal treatment strategies for patients with heterogenous renin-independent aldosteronism.

Disclosure: Y. Yang: None. M. Gao: None. Z. Xia: None. Y. Liu: None. Z. Zhang: None. M. Sun: None. Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism, characterized by upregulation of CYP11B2 gene transcription (encoding aldosterone synthase) and aldosterone biosynthesis in adrenal zona glomerulosa cells. While alterations in metabolic pathways have been reported in APAs, their roles in aldosterone production remain largely undefined. Here, we identified dysregulated propionate metabolism in APAs compared with adjacent zona glomerulosa by bioinformatic analyses using a publicly available dataset (GSE64957). The levels of methylmalonic acid (MMA), a by-product of propionate metabolism, were upregulated in the venous serum from the ipsilateral side of adrenals compared with the contralateral side in patients with APAs (n=15 pairs). This occurred possibly through the upregulation of propionyl-CoA carboxylase alpha (PCCA) in APAs confirmed by immunoblotting and immunohistochemistry. In human adrenocortical cells (HAC15), overexpression or silencing of PCCA caused upregulation or downregulation of CYP11B2 expression and aldosterone production, respectively. MMA treatment in HAC15 cells or C57BL/6 mice also increased CYP11B2 expression and aldosterone production. RNA sequencing revealed an MMA-induced alteration of calcium signaling in HAC15 cells. The elevation of intracellular calcium concentration was later confirmed in MMA-treated cells, and the stimulatory effect of MMA on CYP11B2 expression was reversible by calcium inhibitor nifedipine and calmodulin kinase II inhibitor KN93. Coherently with previous findings, we identified an elevated reactive oxygen species level following MMA treatment. Given the relationship between reactive oxygen species generation and glutathionylation, and the enrichment of the Gene Ontology term “Glutathione binding” in RNA sequencing, we demonstrated that MMA induced glutathionylation in HAC15 cells. Notably, the glutathionylation of L-type calcium channel Cav1.2, which was reported to regulate intracellular calcium influx, was enhanced by MMA. This effect was reversible by the reactive oxygen species scavenger N-acetylcysteine. Mass spectrometry further confirmed that MMA induced the glutathionylation of Cav1.2 at positions Cysteine 106 and Cysteine 621. Collectively, we present a previously uncharacterized dysregulation of propionate metabolism as an important contributor to aldosterone production via MMA-induced Cav1.2 glutathionylation. This pathway represents a potentially valuable target for the development of new therapeutic strategies for primary aldosteronism. Presentation: Saturday, July 12, 2025

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Background Congenital adrenocortical hyperplasia caused by 11β-hydroxylase deficiency (11β-OHD) due to CYP11B1 mutations in 46,XX patients is typically characterized by hyporeninemic hypokalemia hypertension, virilization, precocious pseudopuberty, accelerated skeletal maturation and short stature. Impaired fertility has been reported in the virilizing 11β-OHD form unless properly treated. Case presentation: A 35-year-old female patient with nonclassical 11β-OHD due to novel compound heterozygous mutations (V316M; C262_F264del) in CYP11B1 suffered from menstrual irregularities, infertility, hirsutism and low-renin hypertension with typical hormone profiles including an elevated 11-deoxycorticosterone and testosterone. Her reduced fertility recovered after the initiation of corticosteroid therapy, and conception was later successfully achieved by in vitro fertilization and frozen-thawed embryo transfer. Functional characterization of CYP11B1 V316M and C262_F264del mutations in human adrenocortical cells confirmed 7%-16% of residual enzyme activity (V316M: 11.5% ± 2.4%; C262_F264del: 7.8% ± 2.9%; V316M; C262_F264del: 16.5% ± 0.6%). Conclusions We reported a successful pregnancy in a female patient with nonclassical 11β-OHD due to compound heterozygosity of novel CYP11B1 mutations by in vitro fertilization. The close coordination of care by a multidisciplinary medical team is beneficial for patients with congenital adrenocortical hyperplasia to achieve an accurate diagnosis, proper fertility management and uneventful pregnancy.

Objective: To explore the changes in the health-related quality of life (HRQoL) in patients with primary aldosteronism (PA) after standardized treatment and determine the effects of different variables on the change in the HRQoL of patients. Methods: A total of 116 patients with PA were prospectively included from November 2020 to March 2022. Data were collected at their initial diagnosis and the follow-up after 12 months of treatment, including demographic and clinical data and the scores of the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). The scores of each dimension of SF-36 of patients before and after treatment were compared, and the factors affecting their change in the quality of life were analyzed using multiple linear regression. Results: After standardized treatment, the aldosterone-to-renin ratio (Z =-4.967, P < .001), systolic blood pressure (t = 8.985, P < .001), and diastolic blood pressure (t = 7.233, P < .001) of patients with PA decreased compared with baseline, and hypokalemia was effectively corrected (chi(2) = 69.014, P < .001). In terms of quality of life, 6 of 8 dimensions of SF-36 and the total score of SF-36 significantly improved at 1 -year follow-up compared with baseline (all P < .05). The results of multiple linear regression showed that the improvement in the HRQoL in patients with PA after standardized treatment was correlated with the change in the blood potassium level (P = .007) and systolic blood pressure (P = .003). Conclusion: Correction of hypokalemia and control of diastolic blood pressure are essential factors contributing to the improvement in the HRQoL in patients with PA regardless of the standardized treatment received. (c) 2023 AACE. Published by Elsevier Inc. All rights reserved.

Alcohol consumption has complex effects on diabetes and metabolic disease, but there is widespread heterogeneity within populations and the specific reasons are unclear. Genetic factors may play a role and warrant exploration. The aim of this study was to elucidate genetic variants modulating the impact of alcohol consumption on insulin sensitivity and pancreatic beta cell function within populations presenting normal glucose tolerance (NGT). We recruited 4194 volunteers in Nanjing, 854 in Jurong and an additional 5833 in Nanjing for Discovery cohorts 1 and 2 and a Validation cohort, respectively. We performed an OGTT on all participants, establishing a stringent NGT group, and then assessed insulin sensitivity and beta cell function. Alcohol consumption was categorised as abstinent, light-to-moderate (<210 g per week) or heavy (≥210 g per week). After excluding ineligible individuals, an exploratory genome-wide association study identified potential variants interacting with alcohol consumption in 1862 NGT individuals. These findings were validated in an additional cohort of 2169 NGT individuals. Cox proportional hazard regression was further employed to evaluate the effect of the interaction between the potential variants and alcohol consumption on the risk of type 2 diabetes within the UK Biobank cohort. A significant correlation was observed between drinking levels and insulin sensitivity, accompanied by a consequent inverse relationship with insulin resistance and beta cell insulin secretion after adjusting for confounding factors in NGT individuals. However, no significant associations were noted in the disposition indexes. The interaction of variant rs56221195 with alcohol intake exhibited a pronounced effect on the liver insulin resistance index (LIRI) in the discovery set, corroborated in the validation set (combined p=1.32 × 10−11). Alcohol consumption did not significantly affect LIRI in rs56221195 wild-type (TT) carriers, but a strong negative association emerged in heterozygous (TA) and homozygous (AA) individuals. The rs56221195 variant also significantly interacts with alcohol consumption, influencing the total insulin secretion index INSR120 (the ratio of the AUC of insulin to glucose from 0 to 120 min) (p=2.06 × 10−9) but not disposition index. In the UK Biobank, we found a significant interaction between rs56221195 and alcohol consumption, which was linked to the risk of type 2 diabetes (HR 0.897, p=0.008). Our findings reveal the effects of the interaction of alcohol and rs56221195 on hepatic insulin sensitivity in NGT individuals. It is imperative to weigh potential benefits and detriments thoughtfully when considering alcohol consumption across diverse genetic backgrounds.

Background: Previous studies have reported the direct or indirect relationship between the renin-angiotensinaldosterone system (RAAS) and atrial fibrillation (AF). However, in patients with "apparently" idiopathic AF without possible external influence, whether RAAS is dysregulated at an early stage of AF and its relationship with the recurrence of AF after ablation have not been studied.Methods: This single-center, prospective, case-control study included apparently healthy individuals with AF (the case group) or paroxysmal supraventricular tachycardia (PSVT, the control group) referred for catheter ablation at the same period. The primary outcome was RAAS activation in these two groups. The secondary outcome was the 1-year recurrence of AF after ablation.Results: This study included 51 "apparently" idiopathic AF and 91 patients with PSVT. A greater proportion of patients in the case group had plasma renin activity (PRA) levels < 1 ng/ml/h compared to the control group (25.5 % vs. 7.7 %, P = 0.003). PRA < 1 ng/ml/h was the only factor found to be associated with the diagnose of AF in both the univariate model (odds ratio [OR] 4.11, 95 % confidence interval [CI] 1.52-11.11, P = 0.005) and the model adjusted for age and sex (OR 3.98, 95 % CI 1.20-13.25, P = 0.024). A similar pattern was seen with paroxysmal AF. No significant difference in the components of RAAS was observed between 11 patients with the recurrence of AF and 40 without the recurrence at the 1-year follow-up.Conclusions: This observational study revealed an association between low renin activity and the diagnosis of "apparently" idiopathic AF, particularly paroxysmal AF.

Using the CRISPR/Cas9 genomic editing technology, we constructed a transgenic mouse model to express specific fluorescent protein in pancreatic β cells, which harbor tdTomato exogenous gene downstream of the Ins2 promoter in C57BL/6 J mice. The Ins2-specific single-guide RNA-targeted exon2 was designed for the CRISPR/Cas9 system and Donor vector was constructed at the same time. Then Cas9, sgRNA, and Donor vector were microinjected in vitro into the mouse zygotes that were implanted into pseudo-pregnant mice. We obtained homozygotes through mating heterozygotes, and verified the knockin effect through genotype identification, in vivo imaging, and frozen section. Six F0 mice and stable inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed that the knockin group had no change in the Ins2 exon compared with the control group, while only the base sequence of tdTomato was added and no base mutation occurred. However, in vivo imaging and frozen section did not observe the expression of red fluorescent protein (RFP), and the protein expression of knockin gene tdTomato was negative. As a result, the expressions of tdTomato protein and fluorescence intensity were low and the detection threshold was not reached. In the CRISP/Cas9 technique, the exogenous fragment of IRES connection would affect the transcription level of the preceding gene, which in turn would lead to low-level expression of the downstream gene and affect the effect of gene insertion.

During the pathogenesis of type 1 diabetes (T1D) and type 2 diabetes (T2D), pancreatic islets, especially the β cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet (HFD) and streptozotocin (STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of islet β cells. Cre-LoxP systems were used to generate islet cell type-specific (α, β, or δ) green fluorescent protein (GFP)-labeled mice for genetic lineage tracing, thereinto β-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing (scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled β cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of β cells and some of which transdifferentiated into a or δ cells in both youth- and adulthood-initiated mice while this phenomenon was barely observed in HFD models. β cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into β cells in STZ-treated mice (both youth- and adulthood-initiated). In addition to the re-dedifferentiation of β cells, it is also highly likely that these "α or δ" cells transdifferentiated from pre-existing β cells could also re-trans-differentiate into insulin-producing β cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of β cells. Our findings shed light on how islet β cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis.

This study aimed to develop and validate the accuracy of a clinical-imaging index nomogram in predicting primary aldosteronism (PA) in patients with hypertension. This case-control study enrolled 404 hypertension patients in the First Affiliated Hospital of Nanjing Medical University, China, from April 2017 to September 2021. The patients were randomly divided into the training set (n = 283, 70%) and the validation set (n = 121, 30%). Univariate and multivariate logistic regression analyses were performed to identify independent predictors of PA, which were then used construct a nomogram. The receiver operating characteristic (ROC) curve and calibration plot were drawn to assess the predictive value. The accuracies of our nomogram and other known prediction models were compared using decision curve analyses (DCA). Four significant variables (history of hypokalemia [OR = 2.684, 95% CI: 1.281–5.623, P < 0.001], typical imaging feature [OR = 2.316, 95% CI: 1.166–4.601, P = 0.003], 24 h urine potassium [OR = 0.956, 95% CI: 0.932–0.980, P < 0.001], plasma renin activity [PRA] [OR = 1.423, 95% CI: 1.161–1.744, P < 0.001]) in the multivariate logistic regression analysis were sifted out, and used to build the nomogram. The predictive nomogram yielded an AUC of 0.890 (95% CI, 0.853–0.927) in the training set and 0.860 (95% CI, 0.793–0.927) in the validation set. Predicted and actual probability of PA matched well in the nomogram. Moreover, the DCA showed that the nomogram gained a net benefit in clinical practice in predicting PA when the threshold value was set between 0.1 and 1.0. Our four-variable nomogram was accurate in predicting PA patients and might be introduced into clinical management.

目的:探讨精氨酸代琥珀酸合成酶-1(argininosuccinate synthetase 1,ASS1)对胰岛β细胞增殖与凋亡的影响及机制.方法:利用siRNA和慢病毒载体在胰岛β-TC6细胞中分别敲低和过表达ASS1;5-乙炔基-2′-脱氧尿嘧啶核苷(5-ethynyl-2′-deoxyuridine,EdU)和CCK-8检测细胞增殖能力;Annexin V-PI流式细胞术和原位末端转移酶标记技术(terminal dUTP nick-end labeling assay,TUNEL)检测细胞凋亡水平;Western blot检测B细胞淋巴瘤-2(B-cell leukaemia-2,Bcl-2)蛋白、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、半胱氨酸天冬氨酸酶3(Caspase3)、切割型半胱氨酸天冬氨酸蛋白水解酶-3(cleaved Caspase-3)、凋亡诱导因子(apoptosis inducing factor,AIF)、细胞核增殖抗原(Ki67)和哺乳动物雷帕霉素靶蛋白(mammalian rapamycin target protein,mTOR)的表达水平;RT-qPCR检测AIF、Ki67和mTOR mRNA水平.结果:①与对照相比,敲低ASS1后,胰岛β细胞EdU阳性率和CCK-8细胞增殖活力降低,TUNEL阳性细胞数和AV/PI检测的细胞凋亡率升高.胰岛β细胞内AIF表达量明显升高,Bax/Bcl-2比值下降,Caspase-3活性降低.②过表达ASS1后,TUNEL阳性细胞数和AV/PI检测的细胞凋亡率均较对照组降低,伴随胰岛β细胞内Ki67和mTOR表达量升高.但胰岛β细胞EdU阳性率和CCK-8细胞增殖活力无明显变化.结论:ASS1过表达可能激活mTOR信号通路促进胰岛β细胞增殖;ASS1表达降低时,胰岛β细胞可通过AIF途径启动细胞凋亡.ASS1可能在胰岛β细胞的增殖和凋亡中发挥一定调控作用.