Correction: High frequency of germline recombination in Nestin-Cre transgenic mice crossed with Glucagon-like peptide 1 receptor floxed mice.

[This corrects the article DOI: 10.1371/journal.pone.0296006.].

Single-cell and spatial transcriptomics analysis of human adrenal aging.

OBJECTIVE: The human adrenal cortex comprises three functionally and structurally distinct layers that produce layer-specific steroid hormones. With aging, the human adrenal cortex undergoes functional and structural alteration or "adrenal aging", leading to the unbalanced production of steroid hormones. Given the marked species differences in adrenal biology, the underlying mechanisms of human adrenal aging have not been sufficiently studied. This study was designed to elucidate the mechanisms linking the functional and structural alterations of the human adrenal cortex. METHODS: We conducted single-cell RNA sequencing and spatial transcriptomics analysis of the aged human adrenal cortex. RESULTS: The data of this study suggest that the layer-specific alterations of multiple signaling pathways underlie the abnormal layered structure and layer-specific changes in steroidogenic cells. We also highlighted that macrophages mediate age-related adrenocortical cell inflammation and senescence. CONCLUSIONS: This study is the first detailed analysis of the aged human adrenal cortex at single-cell resolution and helps to elucidate the mechanism of human adrenal aging, thereby leading to a better understanding of the pathophysiology of age-related disorders associated with adrenal aging.

Steroids-producing nodules: a two-layered adrenocortical nodular structure as a precursor lesion of cortisol-producing adenoma.

BACKGROUND: The human adrenal cortex consists of three functionally and structurally distinct layers; zona glomerulosa, zona fasciculata (zF), and zona reticularis (zR), and produces adrenal steroid hormones in a layer-specific manner; aldosterone, cortisol, and adrenal androgens, respectively. Cortisol-producing adenomas (CPAs) occur mostly as a result of somatic mutations associated with the protein kinase A pathway. However, how CPAs develop after adrenocortical cells acquire genetic mutations, remains poorly understood. METHODS: We conducted integrated approaches combining the detailed histopathologic studies with genetic, RNA-sequencing, and spatially resolved transcriptome (SRT) analyses for the adrenal cortices adjacent to human adrenocortical tumours. FINDINGS: Histopathological analysis revealed an adrenocortical nodular structure that exhibits the two-layered zF- and zR-like structure. The nodular structures harbour GNAS somatic mutations, known as a driver mutation of CPAs, and confer cell proliferative and autonomous steroidogenic capacities, which we termed steroids-producing nodules (SPNs). RNA-sequencing coupled with SRT analysis suggests that the expansion of the zF-like structure contributes to the formation of CPAs, whereas the zR-like structure is characterised by a macrophage-mediated immune response. INTERPRETATION: We postulate that CPAs arise from a precursor lesion, SPNs, where two distinct cell populations might contribute differently to adrenocortical tumorigenesis. Our data also provide clues to the molecular mechanisms underlying the layered structures of human adrenocortical tissues. FUNDING: KAKENHI, The Uehara Memorial Foundation, Daiwa Securities Health Foundation, Kaibara Morikazu Medical Science Promotion Foundation, Secom Science and Technology Foundation, ONO Medical Research Foundation, and Japan Foundation for Applied Enzymology.

Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release From Macrophages and Impairs Insulin Secretion.

Pancreatic islet inflammation plays a crucial role in the etiology of type 2 diabetes (T2D). Macrophages residing in pancreatic islets have emerged as key players in islet inflammation. Macrophages express a plethora of innate immune receptors that bind to environmental and metabolic cues and integrate these signals to trigger an inflammatory response that contributes to the development of islet inflammation. One such receptor, Dectin-2, has been identified within pancreatic islets; however, its role in glucose metabolism remains largely unknown. Here we have demonstrated that mice lacking Dectin-2 exhibit local inflammation within islets, along with impaired insulin secretion and ß-cell dysfunction. Our findings indicate that these effects are mediated by proinflammatory cytokines, such as interleukin (IL)-1α and IL-6, which are secreted by macrophages that have acquired an inflammatory phenotype because of the loss of Dectin-2. This study provides novel insights into the mechanisms underlying the role of Dectin-2 in the development of islet inflammation.

High frequency of germline recombination in Nestin-Cre transgenic mice crossed with Glucagon-like peptide 1 receptor floxed mice.

The Cre-loxP strategy for tissue-specific gene inactivation has become a widely employed tool in several research studies. Conversely, inadequate breeding and genotyping without considering the potential for non-specific Cre-recombinase expression may lead to misinterpretations of results. Nestin-Cre transgenic mice, widely used for the selective deletion of genes in neurons, have been observed to have an incidence of Cre-line germline recombination. In this study, we attempted to generate neuron-specific Glucagon-like peptide 1 receptor (Glp1r) knock-out mice by crossing mice harboring the Nestin-Cre transgene with mice harboring the Glp1r gene modified with loxP insertion, in order to elucidate the role of Glp1r signaling in the nervous system. Surprisingly, during this breeding process, we discovered that the null allele emerged in the offspring irrespective of the presence or absence of the Nestin-Cre transgene, with a high probability of occurrence (93.6%). To elucidate the cause of this null allele, we conducted breeding experiments between mice carrying the heterozygous Glp1r null allele but lacking the Nestin-Cre transgene. We confirmed that the null allele was inherited by the offspring independently of the Nestin-Cre transgene. Furthermore, we assessed the gene expression, protein expression, and phenotype of mice carrying the homozygous Glp1r null allele generated from the aforementioned breeding, thereby confirming that the null allele indeed caused a global knock-out of Glp1r. These findings suggest that the null allele in the NestinCre-Glp1r floxed breeding arose due to germline recombination. Moreover, we demonstrated the possibility that germline recombination may occur not only during the spermatogenesis at testis but also during epididymal sperm maturation. The striking frequency of germline recombination in the Nestin-Cre driver underscores the necessity for caution when implementing precise breeding strategies and employing suitable genotyping methods.

Adrenal steroid metabolites and bone status in patients with adrenal incidentalomas and hypercortisolism.

BACKGROUND: Autonomous cortisol secretion (ACS), resulting from cortisol-producing adenomas (CPA), causes endogenous steroid-induced osteoporosis (SIOP). However, the risk of endogenous SIOP cannot be explained by cortisol excess alone, and how other steroid metabolites affect bone status is unclear. METHODS: ACS was diagnosed as serum cortisol ≥1.8 µg/dL after the 1-mg dexamethasone suppression test (DST-cortisol). Using liquid chromatography tandem mass spectrometry, 21 plasma steroid metabolites were measured in 73 patients with ACS and 85 patients with non-functioning adrenal tumors (NFAT). Expression of steroidogenic enzymes and relevant steroid metabolites were analyzed in some of CPA tissues. FINDINGS: Discriminant and principal component analyses distinguished steroid profiles between the ACS and NFAT groups in premenopausal women. Premenopausal women with ACS exhibited higher levels of a mineralocorticoid metabolite, 11-deoxycorticosterone (11-DOC), and lower levels of androgen metabolites, dehydroepiandrosterone-sulfate, and androsterone-glucuronide. In premenopausal women with ACS, DST-cortisol negatively correlated with trabecular bone score (TBS). Additionally, 11-DOC negatively correlated with lumbar spine-bone mineral density, whereas androsterone-glucuronide positively correlated with TBS. The CPA tissues showed increased 11-DOC levels with increased expression of CYP21A2, essential for 11-DOC synthesis. Adrenal non-tumor tissues were atrophied with reduced expression of CYB5A, required for androgen synthesis. INTERPRETATION: This study demonstrates that unbalanced production of adrenal steroid metabolites, derived from both adrenal tumor and non-tumor tissues, contributes to the pathogenesis of endogenous SIOP in premenopausal women with ACS. FUNDING: JSPS KAKENHI, Secom Science and Technology Foundation, Takeda Science Foundation, Japan Foundation for Applied Enzymology, AMED-CREST, JSTA-STEP, JST-Moonshot, and Ono Medical Research Foundation.

Coexistence of bone and vascular disturbances in patients with endogenous glucocorticoid excess.

Purpose: Bone and vascular diseases are considered to share pathogenic mechanisms. Excess glucocorticoids, key regulators of cardiovascular and metabolic homeostasis, may promote both diseases simultaneously. We used endogenous Cushing's syndrome (CS) to investigate whether glucocorticoid excess underlies coexisting bone and vascular diseases. Methods: We included 194 patients with adrenal tumors (ATs): autonomous cortisol secretion (ACS, n = 97) and non-functional AT (n = 97). ACS was further classified into overt CS (n = 17) and subclinical CS (SCS, n = 80). Arterial stiffness was defined as a brachial-ankle pulse wave velocity (baPWV) ≥ 1800 cm/s. Results: Patients with ACS had higher coexistence rates of vertebral fracture and arterial stiffness (23 % vs. 2 %; p < 0.001) and vertebral fracture and abdominal aortic calcification (22 % vs. 1 %; p < 0.001) than those with non-functional AT. In patients with ACS, baPWV was negatively correlated with trabecular bone score (TBS, r = -0.33; p = 0.002), but not with bone mineral density, and vertebral fracture was associated with arterial stiffness in the logistic regression analysis. In the multivariate analysis of variance, the degree of cortisol excess (defined as CS, SCS, and non-functional AT) determined the correlation between TBS and baPWV (partial η2 = 0.07; p < 0.001). In the analysis of covariance, patients with coexisting vertebral fracture and arterial stiffness had higher levels of serum cortisol after the 1-mg dexamethasone suppression test than those without. Conclusion: In endogenous glucocorticoid excess, bone and vascular diseases frequently coexisted, and deteriorated bone quality, not bone loss, was related to arterial stiffness. Thus, glucocorticoid excess may perturb the bone-vascular axis.

Retrograde pyelonephritis and lumbar spondylitis as a result of Salmonella typhi in a type 2 diabetes patient with neurogenic bladder.

We present a case of a 62-year-old diabetic woman with acute pyelonephritis and spondylitis caused by Salmonella typhi. She was admitted to Tokyo Medical Dental University Hospital, Tokyo, Japan, because of unconsciousness and was diagnosed with sepsis by retrograde pyelonephritis as a result of Salmonella typhi. Antibiotics treatment was immediately started; however, she subsequently developed lumbar spondylitis, and long-term conservative treatment with antibiotics and a fixing device were required. This is the first report of a diabetic patient who developed retrograde urinary tract infection with Salmonella typhi, followed by sepsis and spondylitis. The infection could be a result of diabetic neuropathy, presenting neurogenic bladder and hydronephrosis. The patient was successfully treated with antibiotics and became asymptomatic with normal inflammatory marker levels, and no clinical sign of recurrence was observed in the kidney and spine at 4 months.

Increased visceral adiposity with normal weight is associated with the prevalence of non-alcoholic fatty liver disease in Japanese patients with type 2 diabetes.

AIMS/INTRODUCTION: To investigate the impact of increased visceral adiposity with normal weight (OB[-]VA[+]) on the prevalence of non-alcoholic fatty liver disease in patients with type 2 diabetes. MATERIALS AND METHODS: This was a cross-sectional study of 140 Japanese patients with type 2 diabetes (mean age 65 ± 11 year; 44.6% women). Visceral fat area (VFA; cm(2) ) and liver attenuation index (LAI) were assessed by abdominal computed tomography. The patients were divided into four groups by VFA and body mass index (BMI; kg/m(2) ) as follows: BMI <25 kg/m(2) and VFA <100 cm(2) (OB[-]VA[-]), BMI ≥25 kg/m(2) and VFA <100 cm(2) (OB[+]VA[-]), BMI <25 kg/m(2) and VFA ≥100 cm(2) (OB[-]VA[+]), and BMI ≥25 kg/m(2) and VFA ≥100 cm(2) (OB[+]VA[+]). Multivariate linear regression and logistic regression analysis were carried out to determine the impact of OB(-)VA(+) on LAI. RESULTS: In the present study, 25.0% were OB(-)VA(+) patients, where the LAI levels were lower (1.09 ± 0.22) than those in OB(-)VA(-) patients (1.23 ± 0.15), and were equivalent to those in OB(+)VA(+) patients (1.03 ± 0.26). In multivariate linear regression analysis, OB(-)VA(+) was independently associated with LAI (standardized ß-0.212, P = 0.014). In multivariate logistic regression analysis, OB(-)VA(+) was a significant predictor of LAI <0.9 (odds ratio 5.88, 95% confidence interval 1.03-33.52, P = 0.046). CONCLUSIONS: The present study provides evidence that increased visceral adiposity with normal weight is a strong predictor for the prevalence of non-alcoholic fatty liver disease in Japanese patients with type 2 diabetes.

High visceral fat with low subcutaneous fat accumulation as a determinant of atherosclerosis in patients with type 2 diabetes.

BACKGROUND: Abdominal visceral obesity has been reported to be associated with cardiovascular risks than body mass index, waist circumference, and abdominal subcutaneous fat. On the other hand, there is evidence that subcutaneous fat has a beneficial role against cardio-metabolic risks such as diabetes or dyslipidemia. However, little is known regarding the association between high visceral fat with low subcutaneous fat accumulation and the risk for atherosclerosis. METHODS: This study was designed to elucidate whether high visceral fat with low subcutaneous fat accumulation enhances the risk for atherosclerosis in patients with type 2 diabetes. This is a cross-sectional study of 148 patients with type 2 diabetes (mean age 65 ± 12 years; 44.5% female). Visceral fat area (VFA, cm(2)) and subcutaneous fat area (SFA, cm(2)) were assessed by abdominal computed tomography. Carotid intima media thickness (CIMT, mm) measured by ultrasonography was used for the assessment of atherosclerosis. Patients were divided into four groups: SFA < 100 cm(2) and VFA < 100 cm(2) [S(-)V(-)], SFA ≥ 100 cm(2) and VFA < 100 cm(2) [S(+)V(-)], SFA < 100 cm(2) and VFA ≥ 100 cm(2) [S(-)V(+)], and SFA ≥ 100 cm(2) and VFA ≥ 100 cm(2) [S(+)V(+)]. Linear regression analysis with a stepwise procedure was used for the statistical analyses. RESULTS: Among the patients examined, 16.3% were S(-)V(+). Mean (95 % confidence interval) of CIMT adjusting for age and gender were 0.80 (0.69-0.91), 0.86 (0.72-1.01), 1.28 (1.11-1.44) and 0.83 (0.77-0.88) in patients with S(-)V(-), S(+)V(-), S(-)V(+) and S(+)V(+), respectively (p < 0.001). The S(-)V(+) patients exhibited significantly older than S(-)V(-) patients and those with S(+)V(+) and had a highest VFA-SFA ratio (V/S ratio) among the four groups. S(-)V(+) patients were male predominant (100% male), and S(+)V(-) patients showed female predominance (82% female). In multivariate linear regression analysis (Adjusted R(2) = 0.549), S(-)V(+) was significantly associated with CIMT (Standardized ß 0.423, p < 0.001). Notably, S(+)V(+) was inversely associated with CIMT in the multivariate model. CONCLUSIONS: This study provides evidence that high visceral fat with low subcutaneous fat accumulation is an important determinant of carotid atherosclerosis and high subcutaneous fat could be protective against atherosclerosis in patients with type 2 diabetes.

Impact of increased visceral adiposity with normal weight on the progression of arterial stiffness in Japanese patients with type 2 diabetes.

OBJECTIVE: Normal-weight abdominal obesity has been reported to be associated with poor mortality. We aimed to investigate the impact of increased visceral adiposity with normal weight (OB(-)VA(+)) on the progression of arterial stiffness in patients with type 2 diabetes. METHODS: This was a cross-sectional study of 414 patients with type 2 diabetes (mean age 64±12 years; 40.3% female). Visceral fat area (VFA, cm(2)) was measured by a dual bioelectrical impedance analyzer. Arterial stiffness was assessed by brachial-ankle pulse wave velocity (baPWV, cm/s). Patients were divided into four groups by VFA and body mass index (BMI, kg/m(2)) as the following: BMI<25 kg/m(2) and VFA<100 cm(2) (obesity (OB)(-)visceral adiposity (VA)(-)), BMI≥25 kg/m(2) and VFA<100 cm(2) (OB(+)VA(-)), BMI<25 kg/m(2) and VFA≥100 cm(2) (OB(-)VA(+)), and BMI≥25 kg/m(2) and VFA≥100 cm(2) (OB(+)VA(+)). Multivariate linear regression analysis was done to determine the impact of OB(-)VA(+) on arterial stiffness. RESULTS: Among the patients, 7.2% were OB(-)VA(+) with higher baPWV levels (1956±444 cm/s) than those with OB(+)VA(-) (1671±416 cm/s, p=0.014), those with OB(+)VA(+) (1744±317 cm/s, p=0.048), and those with OB(-)VA(-) (1620±397 cm/s, p=0.024). In multivariate linear regression analysis, OB(-)VA(+) remained independently associated with baPWV (standardized ß 0.184, p=0.001). CONCLUSIONS: This study provides evidence for the burden of arterial stiffness in OB(-)VA(+) patients with type 2 diabetes; therefore, evaluation of visceral adiposity is of clinical relevance for the better management of non-obese individuals as well as obese populations.