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Purpose: Optic nerve head (ONH) blood flow decrease without changes in intraocular pressure in a possible rat model of retinal ganglion cell loss by systemic administration of aldosterone. To compare the blood flow in the ONH, using laser speckle flowgraphy (LSFG), in healthy eyes and in eyes with primary aldosteronism (PA). Methods: The ONH tissue area mean blur rate (MT) was evaluated in this single center, retrospective, cross-sectional study using LSFG. In order to compare the MT between PA patients and normal subjects, mixed-effects models were used, with adjustments made for the mean arterial pressure, disc area, and β-peripapillary atrophy (β-PPA) area. Mixed-effects models were also used to analyze the risk factors affecting the MT. Results: This study evaluated a total of 29 eyes of 17 PA patients and 61 eyes of 61 normal subjects. There was a significantly lower MT in PA patients (10.8 ± 0.4) as compared to the normal subjects (12.3 ± 0.3) (P = 0.004). The MT was significantly lower in PA patients (10.8 ± 0.6) even after adjusting for the potential confounding factors when compared to normal subjects (12.3 ± 0.3) (P = 0.046). Multivariate mixed-effects model analysis demonstrated that the MT was significantly associated with the PA and β-PPA. Conclusions: There was a significantly lower ONH blood flow in PA patients as compared to normal subjects. [ABSTRACT FROM AUTHOR]

Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR k cat/ K M for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Ang 1-7 suppressed A-II-stimulated aldosterone production in HAC15 cells. • Ang 1-7 antagonist abrogated Ang 1-7 -mediated inhibition of aldosterone production. • JAK-STAT phosphorylation was inhibited by Ang 1-7 in A-II stimulated HAC15 cells. • STAT signal inhibitors abrogated A-II-stimulated aldosterone production in HAC15. Abstract Angiotensin 1-7 (Ang 1-7), which is a protein cleaved from angiotensin II (A-II), binds to the MAS receptor. Ang 1-7 has been demonstrated to exert protective effects against A-II-mediated cardiac, atherosclerotic, and renal damages. The aims of our study were to demonstrate the inhibitory role of Ang 1-7 in A-II-mediated aldosterone production by interacting with the MAS receptor in human adrenocortical carcinoma (HAC15) cells, and clarify the intracellular signaling mechanisms underlying the inhibition of aldosterone production by Ang 1-7. Ang 1-7 significantly suppressed A-II-stimulated aldosterone production, and partially abrogated A-II-induced upregulation of CYP11B2 expression. Treatment with a selective Ang 1-7 antagonist abrogated Ang 1-7-mediated inhibition of aldosterone production in HAC15 cells. Incubation of A-II-treated HAC15 cells with conditioned medium containing Ang 1-7 was demonstrated to suppress A-II-mediated aldosterone production and CYP11B2 expression. Proteomic analysis showed that Ang 1-7 predominantly inhibited the phosphorylation of JAK-STAT proteins in A-II stimulated HAC15 cells. Treatment of HAC15 cells with a STAT3 inhibitor partially but significantly repressed A-II-mediated aldosterone production by 63.2%. Similarly, treatment with a STAT5 inhibitor significantly abrogated A-II-stimulated aldosterone production in HAC15 cells by 60.7%. In conclusion, we demonstrated that Ang 1-7 negatively regulates A-II-mediated aldosterone production, and the observed inhibition of aldosterone production was associated with JAK/STAT signaling in human adrenal cells. Therefore, activation of Ang 1-7 or stimulation of the MAS receptor, which inhibits aldosterone production, is a promising therapeutic approach for the prevention of cardiovascular events that can directly affect the target organs. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Midnight cortisol levels with high ACTH did not relate with endocrinological findings. • There were fewer patients with hypercortisolism in patients with higher midnight ACTH. • Evaluation of midnight cortisol level was useful in patients with lower midnight ACTH. Abstract Objective Elevated midnight cortisol levels induced by non-suppressed ACTH levels may lead to false-positive results for hypercortisolism in patients with adrenal incidentaloma. We investigated whether plasma ACTH-associated high midnight serum cortisol levels are correlated with other endocrinological findings with respect to hypothalamic-pituitaryadrenal function or hypercortisolism status. Methods Two-hundred-forty-six patients with adrenocortical adenoma were evaluated via measurements of midnight ACTH and cortisol levels, a 1-mg dexamethasone suppression test (DST), and a cosyntropin-releasing hormone (CRH) stimulation test. Patients were divided into four groups according to their midnight plasma ACTH levels. Results The groups with higher midnight ACTH levels had significantly higher basal ACTH levels. A positive relationship was observed between midnight serum cortisol and serum cortisol in the 1-mg DST for all groups; stronger associations were observed in the group with lower midnight ACTH. In the CRH test, peak, delta, and sigma ACTH had significant inverse relationships with midnight cortisol levels in the lowest and second lowest midnight ACTH groups. Patients with midnight cortisol levels >3.5 μg/dL were further divided into two groups according to whether their midnight plasma ACTH levels were below or above 10.0 pg/mL. There were significantly fewer patients with hypercortisolism in the higher ACTH group; midnight serum cortisol levels were associated with hypercortisolism only in the lower ACTH group. Conclusion We demonstrated that midnight ACTH-associated cortisol values were not correlated with other endocrinological findings or hypercortisolism state. Measurement of midnight ACTH levels is important, and careful evaluation is needed for patients with higher midnight ACTH levels. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the methylation levels of G protein-coupled receptor (GPCR) related genes and the effects of methylation on mRNA expression levels in aldosterone-producing adenoma (APA). DNA methylation array and transcriptome analysis were applied in non-functioning adrenocortical adenoma (NFA) and APA. We investigated 192 GPCR-related genes and found hypo-methylation in the promoter region of 66 of these genes in APA. An integration study between microarray and methylation analysis revealed that HTR4 , MC2R , TACR1 , GRM3 , and PTGER1 showed hypo-methylation and up-regulation of mRNA in APA. qPCR analysis showed that HTR4 and PTGER1 expression was 9.3-fold and 6.6-fold higher in APAs than in NFAs, respectively, whereas expression of the other genes was not different between the groups. Methylation of HTR4 and PTGER1 at positions −229 and −666 from the transcription start site, respectively, showed a significant inverse correlation with their mRNA levels. Methylation levels were not associated with KCNJ5 or ATP1A1 mutations in human adrenal samples. We demonstrated an increased incidence of CpG island demethylation of GPCR-related gene in APA. The expression of two receptors, HTR4 and PTGER1 , showed a strong association with DNA methylation. [ABSTRACT FROM AUTHOR]

Yippee-like (YPEL) proteins are thought to be related to cell proliferation because of their structure and location in the cell. The aim of this study was to clarify the effects of YPEL4 on aldosterone production and cell proliferation in the human adrenocortical cell line (HAC15) and aldosterone producing adenoma (APA). Basal aldosterone levels in HAC15 cells over-expressing YPEL4 was higher than those of control HAC15 cells. The positive effects of YPEL4 on cell proliferation were detected by XTT assay and crystal violet staining. YPEL4 levels in 39 human APA were 2.4-fold higher compared to those in 12 non-functional adrenocortical adenomas, and there was a positive relationship between YPEL4 levels and APA diameter (r = 0.316, P < 0.05). In summary, we have demonstrated that YPEL4 stimulates human adrenal cortical cell proliferation, increasing aldosterone production as a consequence. These results in human adrenocortical cells are consistent with the clinical observations with APA in humans. [ABSTRACT FROM AUTHOR]

Under indium Lewis acid catalysis, a nitrogen atom of N-unsubstituted pyrroles was replaced with a nitrogen atom of primary amines, thereby producing N-aryl- and N-alkylpyrroles. This system formally introducing such carbon frameworks to the pyrrole nitrogen atom shows unique selectivity: only the H−N(pyrrolyl) unit undergoes the N-arylation and N-alkylation even in the coexistence of a similar H−N(indolyl) part; and an aryl-halogen bond remains intact. These are clearly different from the typical method depending on the C−N(pyrrolyl) bond-forming reaction with organic halides as substrates. From a viewpoint of pyrrole N-protection-deprotection chemistry, worth noting is that a methyl group on the pyrrole nitrogen atom can be removed, albeit in a formal way. [ABSTRACT FROM AUTHOR]

DNA methylation alteration is tissue-specific and play a pivotal role in regulating gene transcription during cell proliferation and survival. We aimed to detect genes regulated by DNA methylation, and then investigated whether the gene influenced cell proliferation or survival in adrenal cells. DNA methylation and qPCR analyses were performed in nonfunctioning adrenocortical adenoma (NFA, n = 12) and aldosterone-producing adenoma (APA, n = 35) samples. The VDR gene promoter was markedly hypomethylated in APA with ATP1A1 mutation, and the promoter methylation levels showed a significant inverse association with the transcripts in APA. ATP1A1 mutation led to VDR transcription in HAC15 cells, and VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. We demonstrated that APA with ATP1A1 mutation showed entire hypomethylation in the VDR promoter and abundant VDR mRNA and protein expression. VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. Abundant VDR expression would be essential for ATP1A1 mutation-mediated cell proliferation. [Display omitted] • VDR gene promoter was hypomethylated especially in APA with ATP1A1 mutation. • APAs with ATP1A1 mutation had abundant VDR mRNA and protein expression. • ATP1A1 mutation led to VDR transcription in HAC15 cells. • VDR suppression inhibited ATP1A1 mutation-mediated cell proliferation in HAC15 cells. [ABSTRACT FROM AUTHOR]

Objective Absence of a late-night cortisol nadir is a consistent biochemical abnormality in patients with cortisol-producing adenoma. We evaluated the abnormality of late-night urinary free cortisol to creatinine ratio (late-night UFCCR) in patients with subclinical Cushing's syndrome ( SCS). Methods Fifty-eight patients with incidentally detected adrenocortical adenomas [ SCS: 9; nonfunctioning adenoma ( NF): 49] were enrolled as subjects. Values measured in all patients were urinary free cortisol accumulated between 9:00 p.m. and 11:00 p.m. (late-night UFCCR), serum cortisol at 11:00 p.m. (midnight serum cortisol: MSC), serum cortisol after 1-mg overnight dexamethasone suppression test (1 mg- DST) and 24-h urinary free cortisol ( UFC). Results Median late-night UFCCR value in SCS was significantly higher than that in NF ( P < 0·001). Significant correlations were observed between late-night UFCCR and each of serum cortisol after 1 mg- DST and MSC ( r = 0·537, P < 0·001 and r = 0·556, P < 0·001, respectively). There was no significant correlation between serum cortisol after 1 mg- DST and 24-h UFC ( r = 0·211, P = 0·112). In receiver operating characteristic analysis for diagnosis of SCS, the areas under the curves of late-night UFCCR and 24-h UFC were 0·937 (95% confidence interval 0·865-1·008) and 0·726 (0·874-0·999), respectively. Late-night UFCCR cut-off value of 4·9 nmol/μmol Cre showed a sensitivity of 100% and a specificity of 76·6%. Conclusion Patients with SCS showed higher late-night UFCCR values than those with NF. Late-night UFCCR was significantly correlated with autonomous cortisol production findings. Diagnostic performance of late-night UFCCR was superior to 24-h UFC. These results suggest that late-night UFCCR might represent one of the simple and reliable tests for SCS diagnosis. [ABSTRACT FROM AUTHOR]

Summary 1. The mechanisms by which excessive salt causes hypertension involve more than retention of sodium and water by the kidneys and are far from clear. Mineralocorticoids act centrally to increase salt appetite, sympathetic drive and vasopressin release, resulting in hypertension that is prevented by the central infusion of mineralocorticoid receptor (MR) antagonists. The MR has similar affinity for aldosterone and the glucocorticoids corticosterone or cortisol. Specificity is conferred in transport epithelia by the colocalization of the MR with 11β-hydroxysteroid dehydrogenase Type 2. Coexpression also occurs in some neurons, notably those of the nucleus tractus solitarius that are activated by sodium depletion and aldosterone and mediate salt-seeking behaviour. 2. The salt-induced hypertension of the Dahl salt-sensitive rat is mitigated by the central infusion of a mineralocorticoid antagonist even though circulating aldosterone is normal or reduced in salt-sensitive (SS). Contrary to reports that salt appetite in the Dahl salt-sensitive rat is depressed, we found that it is increased compared with that in Spraque-Dawley rats. 3. Extra-adrenal aldosterone synthesis in the brain occurs in minute amounts that could only be relevant locally. Expression of aldosterone synthase mRNA and aldosterone concentrations in the brain of Dahl salt-sensitive rats are increased compared with Spraque-Dawley rats. The central infusion of inhibitors of aldosterone synthesis lowers salt-induced hypertension in the Dahl salt-sensitive rat, suggesting a role for excessive Dahl salt-sensitive synthesis in the brain. Brain MR, particularly those in the paraventricular nuclei, regulate inflammatory processes that are exacerbated by sodium and lead to cardiovascular dysfunction. [ABSTRACT FROM AUTHOR]

Abstract Objective  To identify the molecular mechanisms of bucillamine activity, global gene expression analysis and pathway analysis were conducted using IL-1β-stimulated human fibroblast-like synovial cells (FLS). Methods  Normal human FLS were treated with IL-1β in the presence or absence of 10 and 100 μM bucillamine for 6 h. Total RNA was extracted and global gene expression levels were detected using a 44 k human whole genome array. Data were analyzed using Ingenuity pathway analysis. Results  Numerous pathways were activated by IL-1β stimulation. At both concentrations, bucillamine suppressed nine signal pathways stimulated by IL-1β. Conclusions  Bucillamine effectively inhibited fibroblast growth factor (FGF) signaling and tight junction signaling activated by IL-1β in FLS. Suppression of these signal pathways may correlate with the pharmacologic mechanisms of bucillamine. In particular, the suppression of FGF signaling by bucillamine is remarkable because the activation of FGF signaling may be involved in rheumatoid arthritis pathology. [ABSTRACT FROM AUTHOR]

The intracellular molecular mechanisms underlying the genotype of cortisol-producing adenoma (CPA) have not been fully determined. We analyzed gene expressions in CPA and the human adrenocortical cell line (HAC15 cells) with PRKACA mutation. Clustering analysis using a gene set associated with responses to cAMP revealed the possible differences between PRKACA mutant CPAs and GNAS and CTNNB1 mutant CPAs. The levels of STAR , CYP11A1 , CYP17A1 , CYP21A2, and FDX1 transcripts and cortisol levels per unit area in PRKACA mutant CPAs were significantly higher than those in GNAS mutant CPAs. PRKACA mutations led to an increase in steroidogenic enzyme expression and cortisol production in HAC15 cells. Transcriptome analysis revealed differences between PRKACA mutant CPAs and GNAS and CTNNB1 mutant CPAs. Cortisol production in PRKACA mutant CPAs is increased by the cAMP-PKA signaling pathway-mediated upregulation of steroidogenic enzymes transcription. The intracellular molecular mechanisms underlying these processes would be notably important in PRKACA mutant CPAs. • RNA sequencing analyses showed that PRKACA mutants were separated with others. • Cushing's adenoma with PRKACA mutant had elevated steroidogenic enzyme expression. • Cortisol levels per unit area were high in Cushing's adenoma with PRKACA mutant. • PRKACA mutation increased in steroidogenic enzyme expression in HAC15 cells. • PRKACA mutations led to an increase in cortisol production in HAC15 cells. [ABSTRACT FROM AUTHOR]

A 50-year-old male patient with a 15-year history of hypertension was referred to our hospital for evaluation of bilateral adrenal tumors. No Cushingoid features were observed. Computed tomographic scan showed 10-mm masses in each adrenal gland. Preoperative endocrinological examinations revealed autonomous cortisol and aldosterone secretion in this patient. The results of a subsequent adrenal venous catheterization study were consistent with the presence of a left cortisol-producing tumor and a right aldosterone-producing tumor. A left partial adrenalectomy was performed initially, but cortisol and aldosterone over-secretion persisted. Accordingly, the patient underwent a right adrenalectomy. Pathological examination of the resected specimens, including immunohistochemical analysis, demonstrated that both adenomas possibly produced cortisol and aldosterone. This is an extremely rare case of bilateral adrenal tumors, in which the left adrenocortical tumor produced and secreted cortisol or both cortisol and aldosterone and the right one produced and secreted both aldosterone and cortisol, as confirmed by clinical findings and pathological studies using immunohistochemical analysis. [ABSTRACT FROM AUTHOR]

To guarantee specific tRNA and amino acid pairing, several amino- acyl-tRNA synthetases correct aminoacylation errors by deacylating or "editing" misaminoacylated tRNA. A previously developed variant of Escherichia coil tyrosyl-tRNA synthetase (iodoTyrRS) esterifies or "charges" tRNA[supTyr] with a nonnatural amino acid, 3-iodo-L-tyrosine, and with L-tyrosine less efficiently. In the present study, the editing domain of phenylalanyl-tRNA synthetase (PheRS) was transplanted into iodoTyrRS to edit tyrosyl-tRNA[supTyr] and thereby improve the overall specificity for 3-iodo-L-tyrosine. The β-subunit fragments of the PheRSs from Pyrococcus horikoshii and two bacteria were tested for editing activity. The isolated B314 editing domain of the archaeal PheRS, which was exogenously added to the tyrosylation reaction with iodoTyrRS, efficiently reduced the production of tyrosyl-tRNA[supTyr]. In addition, the transplantation of this domain into iodoTyrRS at the N terminus prevented tyrosyl-tRNA[supTyr] production most strongly among the tested fragments. We next transplanted this archaeal B314 editing domain into iodoTyrRS at several internal positions. Trans- plantation into the connective polypeptide in the Rossmann-fold domain generated a variant that efficiently charges tRNATYr with 3-iodo-L-tyrosine, but hardly produces tyrosyl-tRNA[supTyr]. This variant, iodoTyrRS-ed, was used, together with an amber suppressor derived from tRNA[supTyr], in a wheat germ cell-free translation system and incorporated 3-iodo-i-tyrosine, but not 1-tyrosine, in response to the amber codon. Thus, the editing-domain transplantation achieved unambiguous pairing between the tRNA and the nonnatural amino acid in an expanded genetic code. [ABSTRACT FROM AUTHOR]

Objective Recent studies, both in vitro and in vivo, have indicated that visfatin is one of the inflammatory cytokines, although the relationship between visfatin and insulin resistance remains inconclusive. Accordingly, we assessed the association between visfatin concentrations in serum and those of interleukin-6 (IL-6) and C-reactive protein (CRP), known as markers of systemic inflammation, and also investigated the relationship between these serum concentrations and insulin resistance. Design and method A total of 295 Japanese Americans living in Hawaii (126 men and 169 women, mean age 68·7 ± 14·9 years) were enrolled. The serum levels of visfatin, IL-6 and CRP levels were measured, and homeostasis model assessment for insulin resistance (HOMA-IR) was calculated as a marker of insulin resistance. Results Significant positive correlations were found between serum levels of visfatin and IL-6 or CRP ( r = 0·271, P < 0·001; r = 0·118, P < 0·05, respectively). Multiple regression analysis revealed that correlations between serum levels of visfatin and IL-6 or CRP remained significant after adjustments for age, sex, body mass index, per cent body fat and waist girth. There was no significant trend of the HOMA-IR for the tertiles of serum concentrations of visfatin. On the other hand, a significant trend towards increase of HOMA-IR with increasing tertile of serum concentrations, from the lowest to the highest, was observed for both IL-6 and CRP. The HOMA-IR in subjects with serum concentration of IL-6 or CRP in the highest or intermediate tertiles of IL-6 or CRP were significantly higher than that in subjects in the lowest tertile, even after adjustment for age and sex (IL-6: P < 0·001 and P < 0·001, respectively; CRP: P < 0·001 and P < 0·01, respectively). Conclusion Serum visfatin levels were positively correlated with the serum levels of IL-6 and slightly related with serum levels of CRP, but not with HOMA-IR, in Japanese Americans. Our results indicate that circulating visfatin may reflect inflammation status. [ABSTRACT FROM AUTHOR]

We investigated the effects of marimastat, an inhibitor of TNF-α converting enzyme and matrix metalloproteinases, and anti-TNF-α antibodies on a murine model for sepsis, and on arthritis in human TNF-α transgenic mice. Marimastat (25–200 mg/kg) inhibited lipopolysaccharide (LPS)-induced soluble TNF-α production in mice in a dose-dependent manner. At an oral dose of 200 mg/kg, marimastat almost completely inhibited LPS-induced soluble TNF-α production, but only slightly delayed LPS lethality. On the other hand, anti-TNF-α antibodies completely abolished LPS-induced morbidity. In addition, anti-TNF-α antibodies, but not marimastat (200 mg/kg/day), inhibited the development of arthritis in human TNF-α transgenic mice. These results suggest that cell surface TNF-α may be important in the pathogenesis of murine models for sepsis and arthritis. [Copyright &y& Elsevier]

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA. [ABSTRACT FROM AUTHOR]

Background: Hypertension imposes substantial health and economic burden worldwide. Primary aldosteronism (PA) is one of the most common causes of secondary hypertension, causing cardiovascular events at higher risk compared with essential hypertension. However, the germline genetic contribution to the susceptibility of PA has not been well elucidated. Method: We conducted a genome-wide association analysis of PA in the Japanese population and a cross-ancestry meta-analysis combined with UK Biobank and FinnGen cohorts (816 PA cases and 425 239 controls) to identify genetic variants that contribute to PA susceptibility. We also performed a comparative analysis for the risk of 42 previously established blood pressure–associated variants between PA and hypertension with the adjustment of blood pressure. Results: In the Japanese genome-wide association study, we identified 10 loci that presented suggestive evidence for the association with the PA risk (P <1.0×10−6). In the meta-analysis, we identified 5 genome-wide significant loci (1p13, 7p15, 11p15, 12q24, and 13q12; P <5.0×10−8), including 3 of the suggested loci in the Japanese genome-wide association study. The strongest association was observed at rs3790604 (1p13), an intronic variant of WNT2B (odds ratio, 1.50 [95% CI, 1.33−1.69]; P =5.2×10−11). We further identified 1 nearly genome-wide significant locus (8q24, CYP11B2), which presented a significant association in the gene-based test (P =7.2×10−7). Of interest, all of these loci were known to be associated with blood pressure in previous studies, presumably because of the prevalence of PA among individuals with hypertension. This assumption was supported by the observation that they had a significantly higher risk effect on PA than on hypertension. We also revealed that 66.7% of the previously established blood pressure–associated variants had a higher risk effect for PA than for hypertension. Conclusions: This study demonstrates the genome-wide evidence for a genetic predisposition to PA susceptibility in the cross-ancestry cohorts and its significant contribution to the genetic background of hypertension. The strongest association with the WNT2B variants reinforces the implication of the Wnt/β-catenin pathway in the PA pathogenesis. [ABSTRACT FROM AUTHOR]

This study aimed to develop a machine-learning algorithm to diagnose aldosterone-producing adenoma (APA) for predicting APA probabilities. A retrospective cross-sectional analysis of the Japan Rare/Intractable Adrenal Diseases Study dataset was performed using the nationwide PA registry in Japan comprised of 41 centers. Patients treated between January 2006 and December 2019 were included. Forty-six features at screening and 13 features at confirmatory test were used for model development to calculate APA probability. Seven machine-learning programs were combined to develop the ensemble-learning model (ELM), which was externally validated. The strongest predictive factors for APA were serum potassium (s-K) at first visit, s-K after medication, plasma aldosterone concentration, aldosterone-to-renin ratio, and potassium supplementation dose. The average performance of the screening model had an AUC of 0.899; the confirmatory test model had an AUC of 0.913. In the external validation, the AUC was 0.964 in the screening model using an APA probability of 0.17. The clinical findings at screening predicted the diagnosis of APA with high accuracy. This novel algorithm can support the PA practice in primary care settings and prevent potentially curable APA patients from falling outside the PA diagnostic flowchart. [ABSTRACT FROM AUTHOR]

Brown fat generates heat to protect against cold and obesity. Adrenergic stimulation activates the thermogenic program of brown adipocytes. Although the bioactivity of brown adipose tissue in adult humans had been assumed to very low, several studies using positron emission tomography–computed tomography (PET–CT) have detected bioactive brown adipose tissue in adult humans under cold exposure. In this study, we collected adipose tissues obtained from the perirenal regions of adult patients with pheochromocytoma (PHEO) or non-functioning adrenal tumors (NF). We demonstrated that perirenal brown adipocytes were activated in adult patients with PHEO. These cells had the molecular characteristics of classical brown fat rather than those of beige/brite fat. Expression of brown adipose tissue markers such as uncoupling protein 1 (UCP1) and cell death-inducing DFFA-like effector A (CIDEA) was highly correlated with the amounts of PRD1-BF-1-RIZ1 homologous domain-containing protein-16 (PRDM16) – euchromatic histone-lysine N-methyltransferase 1 (EHMT1) complex, the key transcriptional switch for brown fat development. These results provide novel insights into the reconstruction of human brown adipocytes and their therapeutic application against obesity and its complications such as type 2 diabetes. [ABSTRACT FROM AUTHOR]

Abstract: We recently demonstrated that SA13353 [1-[2-(1-adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea], a novel transient receptor potential vanilloid 1 (TRPV1) agonist, inhibits TNF-α production through the activation of capsaicin-sensitive afferent neurons. In the present study, we investigated the effects of SA13353 on lipopolysaccharide (LPS)-induced cytokine production and a murine model of experimental autoimmune encephalomyelitis (EAE). SA13353 inhibited LPS-induced TNF-α and interleukin (IL)-1β production while augmenting IL-10 production in mice. It also inhibited TNF-α and IL-1β mRNA expression, and increased IL-10 mRNA expression in LPS-treated murine liver. These effects were not observed in TRPV1 KO and sensory denervated mice. Capsaicin and SA13353 increased serum neuropeptide levels, and calcitonin gene-related peptide fragment 8–37 (CGRP8 – 37), a CGRP antagonist, partially blocked the inhibitory effects of capsaicin and SA13353 on LPS-induced TNF-α production. These results suggest that the TPPV1 agonistic effects inhibit TNF-α production, at least partially, via neuropeptide release. SA13353 did not directly affect LPS-induced cytokine production in vitro using RAW264.7 macrophages, which do not express TRPV1. Therefore, we consider SA13353 to be a good tool for the investigation of the value of TRPV1 agonists for the treatment of chronic inflammation. In a murine EAE model, SA13353 attenuated clinical signs and histopathological changes. SA13353 attenuated cytokine levels, including TNF-α, IL-1β, IL-12p40, IL-17, and interferon (IFN)-γ, after proteolipid protein (PLP) immunization. In addition, SA13353 attenuated the increase of IL-17-producing cells. These results suggest that TRPV1 agonists may act as anti-inflammatory and immunomodulatory agents in vivo in certain inflammatory diseases. [Copyright &y& Elsevier]

Summary: We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-l-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-l-tyrosine. The 1.7 Å crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-l-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-l-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Å resolutions by SAD phasing at CuKα and CrKα wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation. [Copyright &y& Elsevier]

We used a dataset from a Japanese nationwide registry of patients with primary aldosteronism, to determine which of the parameters of hyperaldosteronism and blood pressure before or after treatments for primary aldosteronism (i.e., surgical adrenalectomy or a medication treatment) are important in terms of cardiovascular prognosis. We assessed whether plasma aldosterone-to-renin ratio and pulse pressure levels before treatment and 6 months after treatment were associated with composite cardiovascular disease events during the 5-year follow-up period. The cohort included 1987 patients (mean age was 53.2 years, 52.0% were female, 37.2% had undergone surgical treatment, and the remainder had been treated with mineralocorticoid receptor antagonists). In the Cox proportional hazard model, the covariate-adjusted hazard ratio (95% confidence interval) for the composite cardiovascular disease events risk for each one-standard-deviation increase in the aldosterone-to-renin ratio or pulse pressure before treatment, those after treatment, or the duration of hypertension were 1.24 (1.05, 1.48), 0.74 (0.54, 1.02), and 1.07 (0.79, 1.44), 1.43 (1.07, 1.92), and 1.52 (1.19, 1.95), respectively. Patients with a high pre-treatment aldosterone-to-renin ratio of more than 603 and a large post-treatment pulse pressure of more than 49 mmHg showed approximately three-fold higher hazard ratios for cardiovascular events risk compared to those with a lower aldosterone-to-renin ratio and smaller pulse pressure. Higher aldosterone-to-renin ratio before treatments, higher pulse pressure after treatments, and longer duration of hypertension were prognostic factors for cardiovascular diseases. Early intervention may be important for preventing cardiovascular disease among patients with primary aldosteronism. [Display omitted] • Higher pulse pressure after treatments for primary aldosteronism (PA) was a risk factor for cardiovascular diseases (CVD). • Our results suggest that the risk of CVD is associated with how arteriosclerosis progresses before initiating treatment. • Early intervention may be important for preventing CVD among patients with PA. [ABSTRACT FROM AUTHOR]

Pembrolizumab (Keytruda®) is an anti-programmed cell death 1-specific monoclonal antibody that has become the standard second-line chemotherapy for unresectable advanced microsatellite instability-high colorectal cancer. Several immune-related adverse events (irAEs), particularly endocrinopathy, are linked to the administration of pembrolizumab. We report here a case of pembrolizumab-induced isolated adrenocorticotropic hormone deficiency in a patient with metastatic colon cancer. A 65-year-old woman visited our hospital for complaints of fatigue with a recent history of primary resection of cecal mucinous cancer and hepatectomy for liver metastasis 3 years ago. Peritoneal dissemination was detected 2 years after surgery. Several chemotherapeutic regimens of cytotoxic and molecular targeted drugs were administered; however, the metastases progressed gradually. Pembrolizumab monotherapy was started because of resistance to treatment. After 2 cycles of pembrolizumab, the patient was severely fatigued. Laboratory data demonstrated that the cortisol level was extremely low. All the other values were within the normal range. Magnetic resonance imaging indicated no mass in the pituitary gland. From multiple tolerance tests, we diagnosed isolated adrenocorticotropic hormone deficiency caused by pembrolizumab. The patient's symptoms improved promptly with cortisol treatment. An abdominal contrast-enhanced computed tomography scan after 5 cycles of pembrolizumab demonstrated that the size of the peritoneal dissemination remained unchanged. However, her serum level of carcinoembryonic antigen had decreased to normal levels. Endocrine disorders are very rarely seen as irAEs. Careful laboratory data follow-up is required to inhibit the progression of severe endocrine disorders. [ABSTRACT FROM AUTHOR]

Aims/Introduction: The severity of insulin resistance is higher in Japanese‐American people with American lifestyles than in native Japanese people with Japanese lifestyles. Recently, the role of gut microbiota in the control of host metabolic homeostasis and organ physiology has been recognized. In addition, gut microbiota alterations have been suggested to contribute to pathogenesis of insulin resistance. The principle aim of the present study was to evaluate the impact of a Westernized lifestyle on the gut microbiota of Japanese‐Americans versus native Japanese, and its correlation with insulin resistance. Materials and Methods: A total of 14 native Japanese men living in Hiroshima, Japan, and 14 Japanese‐American men living in Los Angeles, USA, were included. A 75‐g oral glucose tolerance test was carried out for all participants to assess their glucose tolerance, and normal glucose tolerance was observed. We compared the insulin response with oral glucose load, the Matsuda Index, and the composition of the gut microbiota between the native Japanese and Japanese‐American men. Results: Japanese‐American men showed higher area under the curve values for serum insulin concentrations during the oral glucose tolerance test and lower Matsuda Index than native Japanese men. Gut microbiota composition of the Japanese‐American men was different; in particular, they showed a relatively lower abundance of Odoribacter than native Japanese men. The ratio between relative abundance of Odoribacter and Matsuda Index was positively correlated between the two groups. Conclusions: Our findings suggest that Westernized lifestyles alter gut microbiota, and its alteration might induce insulin resistance in non‐diabetic Japanese men. [ABSTRACT FROM AUTHOR]

Aims/Introduction: Our previous survey of two Japanese populations, with different lifestyles but identical genetic dispositions, showed that Japanese Americans had different dietary intakes and higher prevalences of obesity and diabetes mellitus, compared with the native Japanese population. The present study examined whether Westernized dietary habits could affect the development of obesity or diabetes. Materials and methods: This study included 765 individuals with normal glucose tolerance at baseline medical examinations (1986 or 1989 in Los Angeles and in 1988 or 1992 in Hawaii) who subsequently completed follow‐up medical examinations several years later. The participants were categorized at baseline as "lean" (576 individuals, body mass index of <25 kg/m2) or "obese" (189 individuals, body mass index of ≥25 kg/m2). Nutrient intakes were analyzed for associations with the development of obesity or diabetes using Cox's proportional hazard model. Results: A total of 41 lean participants developed diabetes, which was not associated with any nutrient intakes (mean follow up 10.8 ± 6.6 years). A total of 36 obese participants developed diabetes, which was positively associated with intakes of animal protein, animal fat and saturated fatty acid (mean follow up 10.7 ± 6.3 years). A total of 85 lean participants became obese, which was positively associated with intakes of simple carbohydrates, sugar and fructose, as well as inversely associated with intakes of vegetable protein and complex carbohydrates (mean follow up 10.4 ± 6.5 years). Conclusions: In the Japanese Americans, different nutrient intakes affected the development of obesity and diabetes. Furthermore, the associations of nutrient intakes with diabetes development varied according to the presence or absence of obesity. [ABSTRACT FROM AUTHOR]

Background: The gut environment has been considered to play a role in the development of nonalcoholic steatohepatitis (NASH). α-glucosidase inhibitors (α-GIs) delay carbohydrate absorption and may change the gut environment. We considered that the protective effect of α-GIs against NASH development is related to changes in the gut environment and thus investigated the effects of miglitol, an α-GI, on NASH development and the gut environment.Methods: Mice were divided into three groups and fed a normal chow diet (NCD), a high-fat high-sucrose diet (HFHSD), or HFHSD plus 0.04% miglitol (HFHSD plus M) for 12 weeks.Results: Insulin resistance developed more in the HFHSD group than in the NCD group, whereas it was suppressed in the HFHSD plus M group. NASH was evaluated histologically, biochemically, and on the basis of messenger RNA expression levels. Miglitol treatment suppressed HFHSD-induced NASH development with the suppression of hepatic Toll-like receptor 4 expression, increased glucagon-like peptide 1 (GLP-1) concentration, and reduced lipopolysaccharide concentration in portal plasma. Regarding the gut environment, the intestinal transit time was shortened and colon inflammation was suppressed in the HFHSD plus M group compared with the HFHSD group. Regarding the gut microbiota, the abundances of Erysipelotrichaceae and Coriobacteriaceae were increased in the HFHSD group compared with the NCD group, whereas the increase was suppressed in the HFHSD plus M group.Conclusions: We demonstrated that miglitol has a protective effect against HFHSD-induced NASH development. The increased GLP-1 secretion and the suppression of endotoxemia, associated with the changes in the gut environment, including the gut microbiota, could contribute to the underlying mechanisms. [ABSTRACT FROM AUTHOR]

Our group previously purified human and rat aldosterone synthase (CYP11B2 and Cyp11b2, respectively) from their adrenals and verified that it is distinct from steroid 11β-hydroxylase (CYP11B1 or Cyp11b1), the cortisol- or corticosterone-synthesizing enzyme. We now describe their distributions immunohistochemically with specific antibodies. In rats, there is layered functional zonation with the Cyp11b2-positive zona glomerulosa (ZG), Cyp11b1-positive zona fasciculata (ZF), and Cyp11b2/Cyp11b1-negative undifferentiated zone between the ZG and ZF. In human infants and children (<12 years old), the functional zonation is similar to that in rats. In adults, the adrenal cortex remodels and subcapsular aldosterone-producing cell clusters (APCCs) replace the continuous ZG layer. We recently reported possible APCC-to-APA transitional lesions (pAATLs) in 2 cases of unilateral multiple adrenocortical micro-nodules. In this review, we present 4 additional cases of primary aldosteronism, from which the extracted adrenals contain pAATLs, with results of next generation sequencing for these lesions. Immunohistochemistry for CYP11B2 and CYP11B1 has become an important tool for the diagnosis of and research on adrenocortical pathological conditions and suggests that APCCs may be the origin of aldosterone-producing adenoma. [ABSTRACT FROM AUTHOR]

Objective: To evaluate whether a Japanese lifestyle during childhood could protect against the future development of obesity-associated metabolic diseases by comparing native Japanese with Japanese-Americans in whom genetic factors are the same. Methods: Study subjects were 516 native Japanese and 781 Japanese-Americans who underwent medical examinations between 2007 and 2010. Japanese-Americans were divided into 444 first-generation immigrants (JA-1), who were born in Japan, and 337 second- or later-generation descendants (JA-2), who were born in the United States. The JA-2 group was then divided into the kibei subgroup (N = 79), who had moved to Japan before the age of 18 years and later returned to the United States, and the non-kibei subgroup (N = 258), who had never lived in Japan. Results: The JA-2 group had the highest percentages of obesity, metabolic syndrome, and type 2 diabetes compared with native Japanese and JA-1. Furthermore, among JA-2, the prevalence of obesity and metabolic syndrome in the kibei subgroup was significantly lower than that in the non-kibei subgroup. The prevalence of diabetes in the kibei subgroup also tended to be lower than in the non-kibei subgroup. Conclusions: The prevalence of obesity and metabolic diseases differed with residence in Japan during childhood among Japanese-Americans. These findings indicate the possibility that Japanese lifestyle during childhood could reduce the future risks for obesity-associated metabolic diseases. [ABSTRACT FROM AUTHOR]

Abstract: Tumor necrosis factor-α (TNF-α) is known to play a crucial role in the pathogenesis of rheumatoid arthritis. In the present study, we demonstrate the effects of SA13353 (1-[2-(1-Adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea), a novel orally active inhibitor of TNF-α production, in animal models, and its mechanism of action on TNF-α production. SA13353 significantly inhibited lipopolysaccharide (LPS)-induced TNF-α production in a dose-dependent manner in rats. Moreover, SA13353 exhibited a binding affinity for the rat vanilloid receptor and increased neuropeptide release from the rat dorsal root ganglion neurons. However, its effects were blocked by pretreatment with the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. The ability of SA13353 and capsaicin to inhibit LPS-induced TNF-α production was eliminated by sensory denervation or capsazepine pretreatment in vivo. Although they inhibited LPS-induced TNF-α production in mice, these effects were not observed in TRPV1 knockout mice. SA13353 provoked the release of neuropeptides without nerve inactivation, even when chronically administered to rats. These results suggest that SA13353 inhibits TNF-α production through activation of capsaicin-sensitive afferent neurons mediated via TRPV1 in vivo. Post-onset treatment of SA13353 strongly reduced the hindpaw swelling and joint destruction associated with collagen-induced arthritis in rats. Thus, SA13353 is expected to be a novel anti-arthritic agent with a unique mechanism of action. [Copyright &y& Elsevier]