心筋細胞におけるステロイドホルモン合成酵素の解明及びその病態生理学的意義

1)ラット心筋におけるアルドステロン産生、及び合成酵素の存在の証明ラット心臓潅流標本を用いて潅流液中にアルドステロンの存在を高速液体クロマトグラフィ(HPLC)、ガスクロマトグラフー質量分析型(GC/MS)を用いて検討を行い,アルドステロンと同等の質量を有するステロイドの存在を確認した.次に心臓をホモジナイズし、[^C]-DOCを加え培養し、培養液をHPLCを用いて分離しアルドステロンの標準ピークに一致する分画を採取し放射活性を測定し、アルドステロンまで変換されるか検討を行なった.心筋におけるアルドステロン合成酵素メッセンジャーRNA(mRNA)の存在を証明するために,心筋からRNAを抽出し逆転写酵素で処置後、アルドステロン合成酵素遺伝子に特異的なプライマーを用いてPCRを行いmRNAの発現を検討した.心筋におけるアルドステロン合成酵素遺伝子mRNAの発現は副腎に比べて100分の1程度と弱かったが,検出可能であった.2)ラット心筋におけるアルドステロン合成調節機構の解明ラットにアンジオテンシン変換酵素阻害薬やアンジオテンシンIIを投与し、また副腎全摘ラットを作製し心臓からのアルドステロンの産生、及び心筋におけるアルドステロン合成酵素活性及びmRNAの発現に対する影響を検討した。心筋におけるアルドステロン合成は副腎同様アンジオテンシンII及びKにより調節をうけ,ACTHの影響は見られなかった.3)脳卒中易発症自然発症高血圧ラット(SHRSP)における検討心筋から産生されたアルドステロンがSHRSPの心肥大進展に及ぼす影響について検討した.心肥大との関連について検討すると,心筋からのアルドステロン産生,合成酵素活性,mRNAの発現は心肥大の進展とともに増加し,心肥大の病態に関与していることが示唆された.次に副腎からのアルドステロンの影響を除外するために,SHRSPの両側副腎摘出を行い,アルドステロン受容体拮抗薬であるスピロノラクトンを投与し心肥大の変化について検討した.両側副腎摘出SHRSPにおいても心肥大の進展がみられたが,スピロノラクトン投与により心肥大の進展は予防できた.これらのことより心筋から産生されるアルドステロンは心肥大の病因に一部関与していることがさらに示唆された.Aldosterone is synthesized in extra-adrenal tissues ; both blood vessels and brain. We undertook this study to determine whether the rat heart produces aldosterone and to investigate the effects of adrenalectomy, angiotensin-converting enzyme (ACE) inhibition, and angiotensin II on aldosterone *nthesis in the heart. To clarify the pathophysiologic role of cardiac aldosterone in the hypertentsive heart, we compared the synthesis of aldosterone in the hearts of stroke-prone spontaneously hypertensive rats (SHRSP) with those in Wistar-Kyoto rats. The effects of the aldosterone antagonist spironolactone on myocardial hypertrophy in adrenalectomized SHRSP were also studied. Isolated rat hearts were perfused for 2 hr, and the perfusate analyzed by high-performance liquid chromatography and mass spectrometry. The activity of aldosterone synthase was estimated by the conversion of [^C] deoxycorticosterone to [^C] aldosterone. The levels of aldosterone synthase gene (CYP11B2) mRNA were determined by competitive polymerase chain reaction. Aldosterone production, the activity of aldosterone synthase, and the expression of CYP11B2 mRNA were increased in hearts from adrenalectomized rats and rats treated with angiotensin II.ACE inhibitors decreased cardiac aldosterone synthesis. Cardiac aldosterone, aldosterone synthase activity and CYP11B2 mRNA levels in hearts from 2- and 4-week-old SHRSP were significantly greater than those of age-matched WKY rats. Spironolactone prevented cardiac hypertrophy in adrenalectomized SHRSP.These results suggest that the rat heart produces aldosterone and endogenous cardiac aldosterone may affect cardiac function and hypertrophy in hypertension in rats.研究課題/領域番号:11838003, 研究期間(年度):1999-200

ホルモン産生腺腫の成因に関する遺伝子学的研究

内分泌臓器によるホルモン産生腫瘍の成因に関して近年遺伝子解析も含め多方面から研究がなされ、家族性甲状腺機能亢進症の一部にG蛋白結合レセプターファミリーであるTSHレセプターの遺伝子異常や、成長ホルモン産生下垂体腺腫の一部にG蛋白の遺伝子異常が報告されているが、多くは不明である。原発性アルドステロン症はアルドステロン過剰産生、高血圧、低カリウム血症を示す疾患であり、心疾患等の合併症を高率に有し、予後不良である。その中でアルドステロン産生腺腫は70%以上を占めるが、その成因は不明である。本研究においてアルドステロン産生腺腫(APA)及び特発性アルドステロン症におけるアルドステロン合成酵素遺伝子(CYP11B2)の異常を検討するためにDNAを抽出し、PCRにて増幅後直接シークエンス法によるDNA解折を行ったが、異常が見られなかった。またグルココルチコイド抑制性アルドステロン症の原因遺伝子であるCYP11Bl/CYP11B2キメラ遺伝子の有無について検討したが、APA、IHAにおいて異常な遺伝子は検出されなかった。アンジオテンシンIIレセプター遺伝子や副腎腺腫を合併する多発性内分泌腺腫症I型の原因遺伝子であるMENIN遺伝子に関してもシークエンスを行ったが、異常が発見できなかった。APA、非機能性副腎腺腫、正常副腎組織におけるCYP11B2mRNAの発現を競合的PCR法にて定量を行うとAPAではCYP11B2mRNAの発現が高値を示した。しかしIHA、APA、非機能性副腎腺腫を有する患者から単球を分離し、単球におけるCYP11B2mRNAの発現を同様に競合的PCR法により検討すると、IHAにおいてCYP11B2mRNAの発現が有意に増加していた。アルドステロン産生腺腫に特異的に発現する遺伝子を解明すべく蛍光ディファレンシャルディスプレイ法を用いて検討した。候補遺伝子をスクリーニングし、ノーザンプロットによるメッセンジャーRNAの発現を検討するとアルドステロン産生腺腫で強く発現し、原因遺伝子の一つである可能性が示唆された。The pathophysiological mechanism of hormone-producing adenoma is unknown. Recently, several researhers habe been reported abnormalities of gene for TSH receptor in thyroid hormone-producing adenoma or mutations of gene for 0-protein in growth hormone-producing pituitary adenoma. Aldosterone-producing adenoma (APA) is characterized by hypertension with excessive production of aldosterone, potassium loss, and suppression of the renin-angiotensin system. Patients with APA are frequently complicated with cardiovascular disease compared with ohter hypertensive patients.I compared activity of aldosterone synthase and expression of CYP11B2 messenger RNA (mRNA) in mononuclear leukocytes (MNL) from patients with IRA to findings in leukocytes from patients with aldosterone-producing adenoma (APA) and normal controls. Levels of CYP11B2 mRNA were determined by competitive PCR.In the same subjects we sought the chimeric CYP1lB1/CYP11B2 that is candidate gene for glucocorticoid-remediable hyperaldos teronism. Southern blot analysis and a long PCR method were used to detect the chimeric gene. Direct sequencing of the CYP11B2 also was performed. No chimeric genes or no mutations in the coding region of the CYP11B2 were found in genomic DNA from these patients. However, both aldosterone synthase activity and CYP11B2 mRNA expression were greater in MNL of patients with IHA than those of patients with APA or controls.I examined the abnormalities of gene for type I angiotensin II receptor (AT1R) or gene for menin, which is the candidate gene for multiple endocrine neoplasia, in aldosteronomas, and found no mutations in both gene gene for AT1R and menin.In order to clarify the candidate gene in aldosteronoma, I isolated highly expressed gene in aldosteronomas compared with normal adrenal tissue or non-functioning adrenal adenomas using a fluorescent differential display method. Northern blot analysis showed that this gene was strongly expressed in aldosteronoma, but not in normal adrenal tissue. These results suggest that this gene is one of the candidate gene for aldosteronoma.研究課題/領域番号:09671036, 研究期間(年度):1997-1998出典：「ホルモン産生腺腫の成因に関する遺伝子学的研究」研究成果報告書　課題番号09671036(KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

ミネラロコルチコイド過剰反応性高血圧症の成因に関する研究

金沢大学医学部1)低レニン血症、低カリウム血症を有する高血圧症患者においてアルドステロン、デオキシコルチコステロン等のミネラロコルチコイド(MC)活性を測定し、原発性及び続発性アルドステロン症を否定した後、最近我々が発見した新しいMCである19-ノルアルドステロンの尿中排泄量を高速液体クロマトグラフィ(HPLC)で分離後、我々が開発した特異的なラジオイムノアッセイにて測定した。2)19-ノルアルドステロンも含めMC活性が高値を示さないMC過剰反応性高血圧症において尿中テトラハイドロコルチゾール、テトラハイドロコルチゾーンをガスクロマトグラフィー質量分析計により測定した。以上の結果よりapparent mineralocorticoid excess syndrome(AME)を疑う症例に関してはゲノムDNAを用いてAMEの原因遺伝子である11β-ハイドロキシステロイド デハイドロゲナーゼ(11β-HSD)II型の遺伝子解析を行った。患者DNAを用いて本酵素に特異的なプライマーを用いてPCRにより増幅し直接DNAシークエンスを行った。3)上記症例のうちAMEも否定的な症例について、尿中11β-HSDII型阻害物質の測定を行った。尿をSep-pakC18で抽出後、HPLCで分離しヒト腎臓マイクロゾーム分画、NAD+、3Hコルチゾールを用いてコルチゾールからコルチゾーンへの変換の阻害率で評価した。標準物質としてグリルレチン酸を用いた。4)尿中11β-HSDII型阻害物質は上記のMC過剰反応性高血圧症において高値を示した。研究課題/領域番号:07671120, 研究期間(年度):1995出典：研究課題「ミネラロコルチコイド過剰反応性高血圧症の成因に関する研究」課題番号07671120（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671120/）を加工して作

Decreased ADP-Ribosyl Cyclase Activity in Peripheral Blood Mononuclear Cells from Diabetic Patients with Nephropathy

Aims/hypothesis. ADP-ribosyl-cyclase activity (ADPRCA) of CD38 and other ectoenzymes mainly generate cyclic adenosine 5’diphosphate-(ADP-) ribose (cADPR) as a second messenger in various mammalian cells, including pancreatic beta cells and peripheral blood mononuclear cells (PBMCs). Since PBMCs contribute to the pathogenesis of diabetic nephropathy, ADPRCA of PBMCs could serve as a clinical prognostic marker for diabetic nephropathy. This study aimed to investigate the connection between ADPRCA in PBMCs and diabetic complications. Methods. PBMCs from 60 diabetic patients (10 for type 1 and 50 for type 2) and 15 nondiabetic controls were fluorometrically measured for ADPRCA based on the conversion of nicotinamide guanine dinucleotide (NGD+) into cyclic GDP-ribose. Results. ADPRCA negatively correlated with the level of HbA1c (P = .040, R2 = .073), although ADPRCA showed no significant correlation with gender, age, BMI, blood pressure, level of fasting plasma glucose and lipid levels, as well as type, duration, or medication of diabetes. Interestingly, patients with nephropathy, but not other complications, presented significantly lower ADPRCA than those without nephropathy (P = .0198) and diabetes (P = .0332). ANCOVA analysis adjusted for HbA1c showed no significant correlation between ADPRCA and nephropathy. However, logistic regression analyses revealed that determinants for nephropathy were systolic blood pressure and ADPRCA, not HbA1c. Conclusion/interpretation. Decreased ADPRCA significantly correlated with diabetic nephropathy. ADPRCA in PBMCs would be an important marker associated with diabetic nephropathy

Changes in aromatase (CYP19) gene promoter usage in non-small cell lung cancer

金沢大学医薬保健研究域医学系In humans, aromatase (CYP19) gene expression is regulated via alternative promoters. Activation of each promoter gives rise to a CYP19 mRNA species with a unique 5′-untranslated region. Inhibition of aromatase has been reported to downregulate lung tumor growth. The genetic basis for CYP19 gene expression and aromatase activity in lung cancer remains poorly understood. We analyzed tissues from 15 patients with non-small cell lung cancer (NSCLC) to evaluate CYP19 promoter usage and promoter-specific aromatase mRNA levels in NSCLC tumor tissues and adjacent non-malignant tissues. CYP19 promoter usage was determined by multiplex RT-PCR and aromatase mRNA levels were measured with real-time RT-PCR. In non-malignant tissues, aromatase mRNA was primarily derived from activation of CYP19 promoter I.4. Although promoter I.4 usage was also dominant in tumor tissues, I.4 activation was significantly lower compared with adjacent non-malignant tissues. Activity of promoters I.3, I.1 and I.7 was significantly higher in tumor tissues compared with non-malignant tissues. In 4 of 15 cases of non-small cell lung cancer, switching from CYP19 promoter I.4 to the alternative promoters II, I.1 or I.7 was observed. In 9 cases, there were significantly higher levels of aromatase mRNA in lung tumor tissues compared with adjacent non-malignant tissues. These findings suggest aberrant activation of alternative CYP19 promoters that may lead to upregulation of local aromatase expression in some cases of NSCLC. Further studies are needed to examine the impact of alternative CYP19 promoter usage on local estrogen levels and lung tumor growth. © 2011 Elsevier Ireland Ltd. All rights reserved

Comparison of eplerenone and spironolactone for the treatment of primary aldosteronism

The mineralocorticoid receptor (MR) is expressed in the kidneys and in adipose tissue, and primary aldosteronism (PA) is associated with metabolic syndrome. This study assessed the effects of MR blockade by eplerenone (EPL) and spironolactone (SPL) on blood pressure (BP) and metabolic factors in patients with PA. Fifty-four patients with PA were treated with one of two MRAs, EPL (25-100 mg daily, n=27) or SPL (12.5-100 mg daily, n=27) for 12 months. Visceral (VAT) and subcutaneous adipose tissue were quantified using CT and FatScan imaging analysis software. Body mass index, homeostasis model assessment-insulin resistance (HOMA-IR), serum creatinine, potassium and lipids, urinary albumin excretion (UAE) and plasma aldosterone concentration (PAC) and plasma renin activity (PRA) were measured before and after treatment. EPL and SPL decreased BP and increased serum potassium levels to similar degrees. PAC and PRA did not differ between the two groups. Although treatment with the MRAs did not change HOMA-IR or serum lipids, they significantly decreased UAE and VAT (P<0.05). These results suggest that EPL and SPL are effective and safe for the treatment of PA. The long-term metabolic and renal effects of these MRAs should be further investigated. © 2016 The Japanese Society of Hypertension. All rights reserved.Embargo Period 6 month

A possible new syndrome with double endocrine tumors in association with an unprecedented type of familial heart-hand syndrome: a case report

<p>Abstract</p> <p>Introduction</p> <p>The combination of a pituitary prolactinoma and an aldosterone-producing adrenal adenoma is extremely rare. To the best of our knowledge, double endocrine tumors in association with heart-hand syndrome have not previously been reported.</p> <p>Case presentation</p> <p>A 21-year-old Japanese woman presented with galactorrhea and decreased visual acuity. A large pituitary adenoma with an increased level of serum prolactin was apparent by computed tomography. She additionally showed mild hypertension (136/90 mmHg) accompanied by hypokalemia. The plasma aldosterone concentration was increased. Computed tomography showed a mass in the right adrenal gland. No other tumors were found despite extensive imaging studies. Physical and radiographic examinations showed skeletal malformations of the hands and feet, including hypoplasia of the first digit in all four limbs. An atrial septal defect was demonstrated by echocardiography. Similar digital and cardiac abnormalities were detected in our patient's father, and a clinical diagnosis of hereditary heart-hand syndrome was made.</p> <p>Conclusion</p> <p>No established heart-hand syndrome was wholly compatible with the family's phenotype. Her father had no obvious endocrine tumors, implying that the parent of transmission determined variable phenotypic expression of the disease: heart-hand syndrome with multiple endocrine tumors from the paternal transmission or no endocrine tumor from the maternal transmission. This suggests that the gene or genes responsible for the disease may be under tissue-specific imprinting control.</p

Impact of serum retinol-binding protein 4 levels on regulation of remnant-like particles triglyceride in type 2 diabetes mellitus

Background. Although retinol-binding protein 4 (RBP4) associates with insulin resistance and remnant-like particles triglyceride (RLP-TG) elevated in the insulin resistant state, few data exist regarding the relationship between RBP4 and RLP-TG. Subjects and Methods. The study included 92 Japanese type 2 diabetic mellitus (T2DM) male patients (age 60.5 ± 13.6 years, body mass index (BMI) 24.7 ± 4.1 kg/m2, waist circumference (WC) 88.4 ± 10.7 cm, and HbA1c (NGSP) 7.2 ± 1.9 %). Patients on medications affecting insulin sensitivity, including fibrates, biguanides, and thiazolidinedione, were excluded. Visceral fat area (VFA) and subcutaneous fat area (SFA) were measured by computed tomography. Results. RBP4 levels showed a significant positive correlation with RLP-TG (r = 0.2544 and P = 0.0056), TG (r = 0.1852 and P = 0.041), RLP-TG/TG (r = 0.23765 and P = 0.0241), and age (r = - 0.2082 and P = 0.0219), although there was no significant correlation with VFA, SFA, adiponectin levels, or homeostasis model of assessment insulin resistance (HOMA-R). Multiple regression analysis revealed that RBP4 was an independent determinant of RLP-TG (P = 0.0193) but was not a determinant of TG. Conclusions. RBP4 correlates positively with serum RLP-TG independent of fat accumulation in T2DM. RBP4 may regulate remnant metabolism independent of glycemic control in T2DM. © 2013 Naoto Yamaaki et al

Clinical Study Impact of Serum Retinol-Binding Protein 4 Levels on Regulation of Remnant-Like Particles Triglyceride in Type 2 Diabetes Mellitus

Background. Although retinol-binding protein 4 (RBP4) associates with insulin resistance and remnant-like particles triglyceride (RLP-TG) elevated in the insulin resistant state, few data exist regarding the relationship between RBP4 and RLP-TG. Subjects and Methods. The study included 92 Japanese type 2 diabetic mellitus (T2DM) male patients (age 60.5 ± 13.6 years, body mass index (BMI) 24.7±4.1 kg/m 2 , waist circumference (WC) 88.4±10.7 cm, and HbA1c (NGSP) 7.2±1.9%). Patients on medications affecting insulin sensitivity, including fibrates, biguanides, and thiazolidinedione, were excluded. Visceral fat area (VFA) and subcutaneous fat area (SFA) were measured by computed tomography. Results. RBP4 levels showed a significant positive correlation with RLP-TG ( = 0.2544 and = 0.0056), TG ( = 0.1852 and = 0.041), RLP-TG/TG ( = 0.23765 and = 0.0241), and age ( = −0.2082 and = 0.0219), although there was no significant correlation with VFA, SFA, adiponectin levels, or homeostasis model of assessment insulin resistance (HOMA-R). Multiple regression analysis revealed that RBP4 was an independent determinant of RLP-TG ( = 0.0193) but was not a determinant of TG. Conclusions. RBP4 correlates positively with serum RLP-TG independent of fat accumulation in T2DM. RBP4 may regulate remnant metabolism independent of glycemic control in T2DM

Diabetes mellitus itself increases cardio- cerebrovascular risk and renal complications in primary aldosteronism

This is a pre-copyedited, author-produced version of an article accepted for publication in The Journal of Clinical Endocrinology & Metabolism following peer review. The version of record Aya Saiki, Michio Otsuki, Daisuke Tamada, Tetsuhiro Kitamura, Iichiro Shimomura, Isao Kurihara, Takamasa Ichijo, Yoshiyu Takeda, Takuyuki Katabami, Mika Tsuiki, Norio Wada, Toshihiko Yanase, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Takanobu Yoshimoto, Ryuji Okamoto, Katsutoshi Takahashi, Hiroki Kobayashi, Kouichi Tamura, Kohei Kamemura, Koichi Yamamoto, Shoichiro Izawa, Miki Kakutani, Masanobu Yamada, Akiyo Tanabe, Mitsuhide Naruse, Diabetes Mellitus Itself Increases Cardio-Cerebrovascular Risk and Renal Complications in Primary Aldosteronism, The Journal of Clinical Endocrinology & Metabolism, Volume 105, Issue 7, July 2020, Pages e2531–e2537 is available online at: https://doi.org/10.1210/clinem/dgaa177