Primary Aldosteronism and Risk of Cardiovascular Outcomes: Genome-Wide Association and Mendelian Randomization Study.

BACKGROUND: Observational studies have reported associations between primary aldosteronism (PA) and cardiovascular outcomes, including coronary artery diseases (CAD), congestive heart failure (CHF), and stroke. However, establishing causality remains a challenge due to the lack of randomized controlled trial data on this topic. We thus aimed to investigate the causal relationship between PA and the risk of developing CAD, CHF, and stroke. METHODS AND RESULTS: Cross-ancestry meta-analysis of genome-wide association studies combining East Asian and European ancestry (1560 PA cases and 742 139 controls) was conducted to identify single-nucleotide variants that are associated with PA. Then, using the identified genetic variants as instrumental variables, we conducted the 2-sample Mendelian randomization analysis to investigate the causal relationship between PA and incident CAD, CHF, and stroke among both East Asian and European ancestry. Summary association results were extracted from large genome-wide association studies consortia. Our cross-ancestry meta-analysis of East Asian and European populations identified 7 genetic loci significantly associated with the risk of PA, for which the genes nearest to the lead variants were CASZ1, WNT2B, HOTTIP, LSP1, TBX3, RXFP2, and NDP. Among the East Asian population, the pooled odds ratio estimates using these 7 genetic instruments of PA were 1.07 (95% CI, 1.03-1.11) for CAD, 1.10 (95% CI, 1.01-1.20) for CHF, and 1.13 (95% CI, 1.09-1.18) for stroke. The results were consistent among the European population. CONCLUSIONS: Our 2-sample Mendelian randomization study revealed that PA had increased risks of CAD, CHF, and stroke. These findings highlight that early and active screening of PA is critical to prevent future cardiovascular events.

Detraining after short-term exercise induces hyperphagia and obesity with fatty liver and brown adipose tissue whitening in young male OLETF rats.

This study examined the effects of exercise and detraining at a young age on fat accumulation in various organs. Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were assigned to either the non-exercise sedentary (OLETF Sed) or exercise groups. The exercise group was subdivided into two groups: exercise between 4 and 12 weeks of age (OLETF Ex) and exercise between 4 and 6 weeks of age followed by non-exercise between 6 and 12 weeks of age (OLETF DT). Body weight was significantly lower in the OLETF Ex group than in the OLETF Sed group at 12 weeks of age. Fat accumulation in the epididymal white adipose tissue, liver, and brown adipose tissue was suppressed in the OLETF Ex group. During the exercise period, body weight and food intake in the OLETF DT group were significantly lower than those in the OLETF Sed group. However, food intake was significantly higher in the OLETF DT group than in the OLETF Sed group after exercise cessation, resulting in extreme obesity with fatty liver and brown adipose tissue whitening. Detraining after early-onset exercise promotes hyperphagia, causing extreme obesity. Overeating should be avoided during detraining periods in cases of exercise cessation at a young age.

Clinical Features of Tachycardia-induced Cardiomyopathy in Patients with Atrial Fibrillation.

Objective Atrial fibrillation (AF) is the most common cause of tachycardia-induced cardiomyopathy (TIC). However, which patients with AF are prone to developing TIC remains unclear. In this study, we investigated the clinical features of AF patients with TIC. Methods This single-center study included 722 patients with AF (average age, 63.1±10.2 years old; 191 women) who underwent radiofrequency catheter ablation. We defined TIC as an initial left ventricular ejection fraction (LVEF) of <40% and a >20% recovery of the LVEF after successful AF ablation and compared the clinical characteristics between the TIC and control groups. Results The proportions of type 2 diabetes (30.5% vs. 14.7%), renal dysfunction (34.2% vs. 23.8%), hypertension (67.1% vs. 54.8%), and persistent AF (62.2% vs. 32.2%) were significantly higher in the TIC group (n=82) than in the control group (n=640). The atrioventricular nodal effective refractory period (AVNERP) (303±72 ms vs. 332±86 ms; p=0.017) was significantly shorter in the TIC group than in the control group. A multivariable analysis found that persistent AF [odds ratio (OR), 3.19; 95% confidence interval (CI), 1.94-5.24], renal dysfunction (OR, 1.87; 95% CI, 1.06-3.32), and type 2 diabetes (OR, 2.30; 95% CI, 1.31-4.05) were significantly associated with TIC. Conclusion Comorbid renal dysfunction and type 2 diabetes were clinical features of AF patients with TIC. Persistent AF, and short AVNERP may be involved in the development of TIC.

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions.

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.

Selective activation of PPARα maintains thermogenic capacity of beige adipocytes.

Beige adipocytes are inducible thermogenic adipocytes used for anti-obesity treatment. Beige adipocytes rapidly lose their thermogenic capacity once external cues are removed. However, long-term administration of stimulants, such as PPARγ and ß-adrenergic receptor agonists, is unsuitable due to various side effects. Here, we reported that PPARα pharmacological activation was the preferred target for maintaining induced beige adipocytes. Pemafibrate used in clinical practice for dyslipidemia was developed as a selective PPARα modulator (SPPARMα). Pemafibrate administration regulated the thermogenic capacity of induced beige adipocytes, repressed body weight gain, and ameliorated impaired glucose tolerance in diet-induced obese mouse models. The transcriptome analysis revealed that the E-twenty-six transcription factor ELK1 acted as a cofactor of PPARα. ELK1 was mobilized to the Ucp1 transcription regulatory region with PPARα and modulated its expression by pemafibrate. These results suggest that selective activation of PPARα by pemafibrate is advantageous to maintain the function of beige adipocytes.

NFIA in adipocytes reciprocally regulates mitochondrial and inflammatory gene program to improve glucose homeostasis.

Adipose tissue is central to regulation of energy homeostasis. Adaptive thermogenesis, which relies on mitochondrial oxidative phosphorylation (Ox-Phos), dissipates energy to counteract obesity. On the other hand, chronic inflammation in adipose tissue is linked to type 2 diabetes and obesity. Here, we show that nuclear factor I-A (NFIA), a transcriptional regulator of brown and beige adipocytes, improves glucose homeostasis by upregulation of Ox-Phos and reciprocal downregulation of inflammation. Mice with transgenic expression of NFIA in adipocytes exhibited improved glucose tolerance and limited weight gain. NFIA up-regulates Ox-Phos and brown-fat-specific genes by enhancer activation that involves facilitated genomic binding of PPARγ. In contrast, NFIA in adipocytes, but not in macrophages, down-regulates proinflammatory cytokine genes to ameliorate adipose tissue inflammation. NFIA binds to regulatory region of the Ccl2 gene, which encodes proinflammatory cytokine MCP-1 (monocyte chemoattractant protein-1), to down-regulate its transcription. CCL2 expression was negatively correlated with NFIA expression in human adipose tissue. These results reveal the beneficial effect of NFIA on glucose and body weight homeostasis and also highlight previously unappreciated role of NFIA in suppressing adipose tissue inflammation.

NFIA determines the cis-effect of genetic variation on Ucp1 expression in murine thermogenic adipocytes.

Thermogenic brown and beige adipocytes counteract obesity by enhancing energy dissipation via uncoupling protein-1 (Ucp1). However, the effect of genetic variation on these cells, a major source of disease susceptibility, has been less well studied. Here we examined beige adipocytes from obesity-prone C57BL/6J (B6) and obesity-resistant 129X1/SvJ (129) mouse strains and identified a cis-regulatory variant rs47238345 that is responsible for differential Ucp1 expression. The alternative T allele of rs47238345 at the Ucp1 -12kb enhancer in 129 facilitates the allele-specific binding of nuclear factor I-A (NFIA) to mediate allele-specific enhancer-promoter interaction and Ucp1 transcription. Furthermore, CRISPR-Cas9/Cpf1-mediated single nucleotide polymorphism (SNP) editing of rs47238345 resulted in increased Ucp1 expression. We also identified Lim homeobox protein 8 (Lhx8), whose expression is higher in 129 than in B6, as a trans-acting regulator of Ucp1 in mice and humans. These results demonstrate the cis- and trans-acting effects of genetic variation on Ucp1 expression that underlie phenotypic diversity.

Effects of long-term childhood exercise and detraining on lipid accumulation in metabolic-related organs.

The preventive effects of regular exercise on obesity-related health problems are carried over to the non-exercise detraining period, even when physical activity decreases with aging. However, it remains unknown whether regular childhood exercises can be carried over to adulthood. Therefore, this study aimed to investigate the effects of long-term childhood exercise and detraining on lipid accumulation in organs to prevent obesity in adulthood. Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used as obese animals. OLETF rats were allocated into sedentary and exercise groups: exercise from 4- to 12-week-old and detraining from 12- to 20-week-old. At 12-week-old immediately after the exercise period, regular exercise completely inhibited hyperphagia, obesity, enlarged pancreatic islets, lipid accumulation and lobular inflammation in the liver, hypertrophied adipocytes in the white adipose tissue (WAT), and brown adipose tissue (BAT) whitening in OLETF rats. Additionally, exercise attenuated the decrease in the ratio of muscle wet weight to body weight associated with obesity. Decreased food consumption was maintained during the detraining period, which inhibited obesity and diabetes at 20-week-old after the detraining period. Histologically, childhood exercise inhibited the enlargement of pancreatic islets after the detraining period. In addition, inhibition of lipid accumulation was completely maintained in the WAT and BAT after the detraining period. However, the effectiveness was only partially successful in lipid accumulation and inflammation in the liver. The ratio of muscle wet weight to body weight was maintained after detraining. In conclusion, early long-term regular exercise effectively prevents obesity and diabetes in childhood, and its effectiveness can be tracked later in life. The present study suggests the importance of exercise during childhood and adolescence to inhibit hyperphagia-induced lipid accumulation in metabolic-related organs in adulthood despite exercise cessation.

Masticatory dysfunction in patients with diabetic neuropathy: A cross-sectional study.

INTRODUCTION: Chewing well is essential for successful diet therapy and control of blood glucose level in patients with diabetes. In addition, long-term hyperglycemia is a risk factor for microvascular complications, which are the main cause of morbidity and mortality in these patients. Hence, it is plausible that masticatory disorder may be relevant to diabetic microvascular complications which is caused by long-term hyperglycemia. The aim of this study was to investigate whether masticatory disorders are relevant to diabetic microvascular complications. METHODS: This cross-sectional study included 172 patients with type 2 diabetes who underwent educational hospitalization in the Department of Endocrinology and Diabetic Medicine, Hiroshima University Hospital, from April 2016 to March 2020. Masticatory efficiency was determined quantitatively by using the GLUCO SENSOR GS-Ⅱ. Multivariable linear regression models were constructed to examine which factors were related to masticatory efficiency. Statistical significance was defined as a two-sided p value of < 0.05. RESULTS: According to the bivariable analysis, masticatory efficiency was significantly correlated with duration of diabetes (p = 0. 049), number of remaining teeth (p < 0.0001), the number of moving teeth (p = 0.007) and condition of diabetic neuropathy (p < 0.0001). Moreover, the number of remaining teeth (p < 0.0001) and diabetic neuropathy (p = 0.007) remained significantly correlated with masticatory efficiency in the multivariable analysis. CONCLUSIONS: For the first time, we demonstrated that patients with type 2 diabetes who developed diabetic neuropathy had significantly reduced masticatory efficiency. Effective mastication is an important factor in successful diet therapy for diabetes. To prevent the progression of diabetic complications, especially in patients with diabetic neuropathy, it may be necessary to combine individualized therapies from dentists and nutritionists with consideration for the level of masticatory dysfunction.

Association of DNA methylation with steroidogenic enzymes in Cushing's adenoma.

DNA methylation and demethylation regulate the transcription of genes. DNA methylation-associated gene expression of adrenal steroidogenic enzymes may regulate cortisol production in cortisol-producing adenoma (CPA). We aimed to determine the DNA methylation levels of all genes encoding steroidogenic enzymes involved in CPA. Additionally, the aims were to clarify the DNA methylation-associated gene expression and evaluate the difference of CPA genotype from others using DNA methylation data. Twenty-five adrenal CPA and six nonfunctioning adrenocortical adenoma (NFA) samples were analyzed. RNA sequencing and DNA methylation array were performed. The methylation levels at 118 methylation sites of the genes were investigated, and their methylation and mRNA levels were subsequently integrated. Among all the steroidogenic enzyme genes studied, CYP17A1 gene was mainly found to be hypomethylated in CPA compared to that in NFA, and the Benjamini-Hochberg procedure demonstrated that methylation levels at two sites in the CYP17A1 gene body were statistically significant. PRKACA mutant CPAs predominantly exhibited hypomethylation of CYP17A1 gene compared with the GNAS mutant CPAs. Inverse associations between CYP17A1 methylation in three regions of the gene body and its mRNA levels were observed in the NFAs and CPAs. In applying clustering analysis using CYP17A1 methylation and mRNA levels, CPAs with PRKACA mutation were differentiated from NFAs and CPAs with a GNAS mutation. We demonstrated that CPAs exhibited hypomethylation of the CYP17A1 gene body in CPA, especially in the PRKACA mutant CPAs. Methylation of CYP17A1 gene may influence its transcription levels.

Hypomethylation associated vitamin D receptor expression in ATP1A1 mutant aldosterone-producing adenoma.

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.

Dietary Vitamins A, C, and Potassium Intake Is Associated With Narrower Retinal Venular Caliber.

INTRODUCTION: The retinal vasculature, a surrogate for the systemic microvasculature, can be observed non-invasively, providing an opportunity to examine the effects of modifiable factors, such as nutrient intake, on microcirculation. We aimed to investigate the possible associations of dietary nutrient intake with the retinal vessel caliber. METHODS: In this cross-sectional study, a total of 584 participants in a medical survey of Japanese descendants living in Los Angeles in 2015 underwent a dietary assessment, fundus photographic examination, and comprehensive physical and blood examinations. Retinal vessel caliber was measured using fundus photographs with a semi-automated computer system and summarized as central retinal artery and vein equivalents (CRAE and CRVE). The association between dietary nutrient intake and retinal vessel caliber was analyzed using a multivariate linear regression model adjusted for two models including potential confounders. The first model was adjusted for age and sex. The second model was adjusted for age, sex, smoking status, body mass index, hypertension, diabetes, dyslipidemia, history of coronary heart disease, and history of stroke. RESULTS: After adjustment of potential confounders, compared to the quartile with the lowest intake, the difference in CRVE for the highest quartile was -5.33 µm [95% confidence interval (CI): -9.91 to -0.76, P for trend = 0.02] for vitamin A, -4.93 µm (95% CI: -9.54 to -0.32, P for trend = 0.02) for vitamin C and -3.90 µm (95% CI: -8.48 to 0.69, P for trend = 0.04) for potassium. CONCLUSIONS: A significant association was observed between higher vitamins A, C and potassium intakes and narrower retinal venular caliber.

Association of Dietary Nutrient Intake with Early Age-Related Macular Degeneration in Japanese-Americans.

Lifestyle factors may be associated with the development of age-related macular degeneration (AMD), in addition to demographic and genetic factors. The purpose of this cross-sectional study is to elucidate the association between nutrient intake and AMD in the Japanese-American population living in Los Angeles. We conducted a medical survey of Japanese immigrants and their descendants living in Los Angeles, including interviews on dietary habits, fundus photography, and physical examinations. Participants were classified into early AMD and control groups on the basis of fundus photographic findings. Consequently, among the 555 participants, 111 (20.0%) were diagnosed with early AMD. There were no late-stage AMD participants. Multivariate logistic regression analysis showed that the intake of animal fat and saturated fatty acids (SFA) was positively associated with early AMD (p for trend = 0.01 for animal fat, p for trend = 0.02 for SFA), and the intake of vegetable fat, total carbohydrate, simple carbohydrate, sugar, and fructose was inversely associated with early AMD (p for trend = 0.04 for vegetable fat, p for trend = 0.046 for carbohydrate, p for trend = 0.03 for simple carbohydrate, p for trend = 0.046 for sugar, p for trend = 0.02). Our findings suggest that excessive animal fat and SFA intake increases the risk for early AMD in Japanese-Americans whose lifestyles have been westernized.

ATP1A1 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation.

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.

Genotype-specific cortisol production associated with Cushing's syndrome adenoma with PRKACA mutations.

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.

Update on Genetics of Primary Aldosteronism.

Primary aldosteronism (PA) is the most common form of secondary hypertension, with a prevalence of 5-10% among patients with hypertension. PA is mainly classified into two subtypes: aldosterone-producing adenoma (APA) and bilateral idiopathic hyperaldosteronism. Recent developments in genetic analysis have facilitated the discovery of mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, CLCN2, and CTNNB1 in sporadic or familial forms of PA in the last decade. These findings have greatly advanced our understanding of the mechanism of excess aldosterone synthesis, particularly in APA. Most of the causative genes encode ion channels or pumps, and their mutations lead to depolarization of the cell membrane due to impairment of ion transport. Depolarization activates voltage-gated Ca2+ channels and intracellular calcium signaling and promotes the transcription of aldosterone synthase, resulting in overproduction of aldosterone. In this article, we review recent findings on the genetic and molecular mechanisms of PA.

Variation in plasma glucagon levels according to obesity status in Japanese Americans with normal glucose tolerance.

Japanese Americans living in the United States are genetically identical to Japanese people, but have undergone a rapid and intense westernization of their lifestyle. This study investigated variability in glucagon secretion after glucose loading among Japanese Americans with normal glucose tolerance (NGT) according to obesity status. The 75-g oral glucose tolerance test (OGTT) was performed for 138 Japanese Americans (aged 40-75 years) living in Los Angeles. Plasma glucagon levels measured using the sandwich enzyme-linked immunosorbent assay were compared according to body mass index (BMI) categories among 119 individuals with NGT. The individuals were classified into three categories according to their BMI values: <22 kg/m2 (n = 37), 22-24.9 kg/m2 (n = 46), and ≥25 kg/m2 (n = 36). Fasting plasma glucagon levels and glucagon-area under the curve levels during the OGTT were the highest in the BMI ≥25 kg/m2 group. Fasting glucagon levels were correlated with BMI (r = 0.399, p < 0.001), fasting insulin levels (r = 0.275, p = 0.003) and the homeostasis model assessment-insulin resistance (r = 0.262, p = 0.004). In conclusion, our findings suggest that fasting hyperglucagonemia is associated with obesity and insulin resistance even during the NGT stage in the Japanese American population.

Angiotensin-converting enzyme 2 expression is not induced by the renin-angiotensin system in the lung.

Pulmonary expression of angiotensin-converting enzyme 2, which is a receptor of severe acute respiratory syndrome coronavirus 2, is not regulated by angiotensin II or renin-angiotensin system inhibitors #COVID19 https://bit.ly/3fkopuO.

Effect of the use of passive body trunk exercise equipment on oxygen consumption and self-efficacy for carrying out exercise in patients with type 2 diabetes.

AIMS/INTRODUCTION: Considering the difficulty in inculcating the habit of exercise among patients with type 2 diabetes, devising an easily maintained means of exercise is preferable. Passive body trunk exercise equipment (PBTE) developed for home use might solve several problems related to exercise therapy, both for patients and clinical staff involved in diabetes treatment; however, its efficacy as a therapeutic exercise device for patients with diabetes has not been ascertained. The purpose of this study was to measure the exercise intensity and self-efficacy of PBTE, and to determine whether PBTE is a useful tool for exercise therapy. MATERIALS AND METHODS: The participants were 20 patients with type 2 diabetes, and the duration of exercise using the PBTE was set to 10 min. Oxygen consumption during exercise was measured, and self-efficacy for continuing to exercise using the PBTE and for walking was evaluated after completion of the study. RESULTS: The average exercise intensity using the PBTE was 1.7 metabolic equivalents, whereas the maximum exercise intensity was an average of 2.0 metabolic equivalents; the reported self-efficacy for continuing to exercise using the PBTE was significantly higher than for walking. CONCLUSIONS: Exercise intensity using the PBTE is similar to low-intensity walking, and thus, it might be a useful therapeutic exercise device for patients with type 2 diabetes. Furthermore, it could be an effective exercise device for diabetes patients who do not have regular exercise habits, especially with reduced motor function or lower leg muscle strength.

Endoplasmic Reticulum Chaperone Calmegin Is Upregulated in Aldosterone-Producing Adenoma and Associates With Aldosterone Production.

The endoplasmic reticulum (ER) plays a pivotal role in syntheses of proteins and steroid hormones and regulation of intracellular Ca2+ level. We aimed to investigate ER-associated genes in aldosterone-producing adenomas (APAs) and clarify their effect on aldosterone production. Microarray analysis targeting 288 ER-associated genes was conducted using nonfunctioning adrenocortical adenomas (n=5) and APAs (n=19). Immunohistochemistry and quantitative polymerase chain reaction analyses were performed with 13 nonfunctioning adrenocortical adenoma and 48 APA samples. Functional studies were performed with human adrenocortical carcinoma (HAC15) cells, some of which were genetically modified using lentiviruses. The ER chaperone calmegin (CLGN) was the most highly expressed ER-associated gene in APAs relative to nonfunctioning adrenocortical adenomas. Analysis with quantitative polymerase chain reaction revealed CLGN to be 9.5-fold upregulated in APAs relative to nonfunctioning adrenocortical adenomas. There were no differences among different APA genotypes affecting aldosterone production. Immunohistochemistry analysis revealed that CLGN was strongly expressed in APAs and aldosterone-producing cell clusters. Angiotensin II stimulation or KCNJ5 T158A overexpression in HAC15 cells did not affect CLGN mRNA levels. CLGN overexpression in HAC15 cells increased aldosterone levels but did not stimulate CYP11B2 mRNA levels. Pathway and gene ontology analyses using RNA sequencing results showed that tRNA aminoacyl metabolism was the most enriched pathway in CLGN-overexpressing cells. CYP11B2 (aldosterone synthase) and HSD3B2 (3 beta-hydroxysteroid dehydrogenase/delta 5->4-isomerase type 2) protein expression were more abundant in CLGN-overexpressing cells. CLGN knockdown using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method in HAC15 cells that carry the KCNJ5 mutation did not affect aldosterone production. To summarize, CLGN was upregulated and associated with aldosterone production via translational regulation of CYP11B2 in APAs.