Takako Araki

Vitamin D regulates steroidogenesis and insulin-like growth factor binding protein-1 (IGFBP-1) production in human ovarian cells.

Vitamin D Receptor (VDR) is expressed in both animal and human ovarian tissue, however, the role of vitamin D in human ovarian steroidogenesis is unknown. Cultured human ovarian cells were incubated in tissue culture medium supplemented with appropriate substrates, with or without 50 pM-150 pM or 50 nM-150 nM of 1,25-(OH)2D3, and in the presence or absence of insulin. Progesterone, testosterone, estrone, estradiol, and IGFBP-1 concentrations in conditioned tissue culture medium were measured. Vitamin D receptor was present in human ovarian cells. 1,25-(OH)2D3 stimulated progesterone production by 13% (p<0.001), estradiol production by 9% (p<0.02), and estrone production by 21% (p<0.002). Insulin and 1,25-(OH)2D3 acted synergistically to increase estradiol production by 60% (p<0.005). 1,25-(OH)2D3 alone stimulated IGFBP-1 production by 24% (p<0.001), however, in the presence of insulin, 1,25-(OH)2D3 enhanced insulin-induced inhibition of IGFBP-1 production by 13% (p<0.009). Vitamin D stimulates ovarian steroidogenesis and IGFBP-1 production in human ovarian cells likely acting via vitamin D receptor. Insulin and vitamin D synergistically stimulate estradiol production. Vitamin D also enhances inhibitory effect of insulin on IGFBP-1 production.

Vitamin D Intoxication with Severe Hypercalcemia due to Manufacturing and Labeling Errors of Two Dietary Supplements Made in the United States

CONTEXT More than 50% of Americans use dietary supplements, and 60-70% fail to report this use to their physicians. Intoxication from vitamin D supplements has been rarely reported but may now occur more frequently. This may be attributable to an increase in vitamin D supplement intake due to the findings that deficiency is common and has been associated with a number of disease states. OBJECTIVE We report two cases of vitamin D intoxication with dietary supplements made in the United States caused by manufacturing and labeling errors. METHODS Case histories were obtained, and serial laboratory data (calcium and vitamin D metabolites) were measured. Each dietary supplement was analyzed by UV spectrophotometry followed by HPLC. RESULTS In both cases, repetitive inquiries were required to elicit the use of dietary supplements. Because of significant manufacturer errors and a labeling error, patients had been consuming more than 1000 times the recommended daily dose of vitamin D(3). Hypercalcemia is directly proportional to serum 25-hydroxyvitamin D [25(OH)D] but not 1,25-dihydroxyvitamin D levels. It took approximately 1 yr to normalize 25(OH)D levels. However, once 25(OH)D levels decreased below 400 ng/ml, both patients became normocalcemic and asymptomatic without long-term sequelae. CONCLUSIONS Although rare, vitamin D intoxication should be considered in the differential diagnosis of hypercalcemia. Patients should be asked whether they are using dietary supplements, and serial questioning may be required because patients may not consider these supplements to be potential health risks. Errors in the manufacturing and labeling of dietary supplements made in the United States may place individuals at increased risks for side effects.

Cyclin E-Mediated Human Proopiomelanocortin Regulation as a Therapeutic Target for Cushing Disease.

CONTEXT Cushing disease, due to pituitary corticotroph tumor ACTH hypersecretion, drives excess adrenal cortisol production with adverse morbidity and mortality. Loss of glucocorticoid negative feedback on the hypothalamic-pituitary-adrenal axis leads to autonomous transcription of the corticotroph precursor hormone proopiomelanocortin (POMC), consequent ACTH overproduction, and adrenal hypercortisolism. We previously reported that R-roscovitine (CYC202, seliciclib), a 2,6,9-trisubstituted purine analog, suppresses cyclin-dependent-kinase 2/cyclin E and inhibits ACTH in mice and zebrafish. We hypothesized that intrapituitary cyclin E signaling regulates corticotroph tumor POMC transcription independently of cell cycle progression. The aim was to investigate whether R-roscovitine inhibits human ACTH in corticotroph tumors by targeting the cyclin-dependent kinase 2/cyclin E signaling pathway. METHODS Primary cell cultures of surgically resected human corticotroph tumors were treated with or without R-roscovitine, ACTH measured by RIA and quantitative PCR, and/or Western blot analysis performed to investigate ACTH and lineage-specific transcription factors. Cyclin E and E2F transcription factor 1 (E2F1) small interfering RNA (siRNA) transfection was performed in murine corticotroph tumor AtT20 cells to elucidate mechanisms for drug action. POMC gene promoter activity in response to R-roscovitine treatment was analyzed using luciferase reporter and chromatin immunoprecipitation assays. RESULTS R-roscovitine inhibits human corticotroph tumor POMC and Tpit/Tbx19 transcription with decreased ACTH expression. Cyclin E and E2F1 exhibit reciprocal positive regulation in corticotroph tumors. R-roscovitine disrupts E2F1 binding to the POMC gene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways. CONCLUSION R-roscovitine inhibits human pituitary corticotroph tumor ACTH by targeting the cyclin E/E2F1 pathway. Pituitary cyclin E/E2F1 signaling is a previously unappreciated molecular mechanism underlying neuroendocrine regulation of the hypothalamic-pituitary-adrenal axis, providing a subcellular therapeutic target for small molecule cyclin-dependent kinase 2 inhibitors of pituitary ACTH-dependent hypercortisolism, ie, Cushing disease.

Rosiglitazone and pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase

The effects of rosiglitazone or pioglitazone (thiazolidinediones, TZDs) on estrogen production and aromatase activity in human ovarian cells were examined. Human granulosa cells were incubated in the tissue culture medium supplemented with androstenedione or testosterone, with or without insulin, TZDs, or type 1 17β-hydroxysteroid-dehydrogenase (17β-HSD) inhibitor. Estrogen concentrations in the conditioned medium, aromatase mRNA and protein expression in the cells and androgen substrate binding to aromatase were measured. With androstenedione as substrate, rosiglitazone or pioglitazone inhibited estrone production by up to 22% (p<0.012) while type 1 17β-HSD inhibitor enhanced this effect of rosiglitazone or pioglitazone by 37% (p<0.001) and by 67% (p<0.001), respectively. With testosterone as substrate, rosiglitazone or pioglitazone inhibited estradiol production by 32% (p<0.001). With (3)H-testosterone as substrate, rosiglitazone or pioglitazone inhibited the (3)H-tritiated water release by the cultured cells by 45% and 35%, respectively, thus directly demonstrating inhibition of aromatase. Rosiglitazone or pioglitazone, however, had no significant effect on aromatase mRNA or protein expression. Rosiglitazone or pioglitazone inhibited (125)I-androstenedione and (125)I-testosterone binding to aromatase by 38% (p<0.001). It was concluded that rosiglitazone or pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase.

Growth hormone is permissive for neoplastic colon growth.

Significance Growth hormone (GH) excess in acromegaly is associated with increased colon polyps and cancer, whereas short-stature humans harboring a GH receptor mutation do not develop cancer. Administration of a GH receptor blocker in acromegaly patients induced colon p53. In contrast, p53 is suppressed by GH in colon cells, in vivo in colon tissue, and in induced pluripotent stem cell-derived intestinal organoids. GH excess leads to cell survival with downregulated adenomatous polyposis coli, nuclear β-catenin accumulation, and increased epithelial–mesenchymal transition factors. Because locally expressed GH is abundant in conditions predisposing to colon cancer, GH appears to be a molecular component of the milieu permissive for neoplastic colon growth. These results explain the protective effects of GH deficiency against development of neoplasms. Growth hormone (GH) excess in acromegaly is associated with increased precancerous colon polyps and soft tissue adenomas, whereas short-stature humans harboring an inactivating GH receptor mutation do not develop cancer. We show that locally expressed colon GH is abundant in conditions predisposing to colon cancer and in colon adenocarcinoma-associated stromal fibroblasts. Administration of a GH receptor (GHR) blocker in acromegaly patients induced colon p53 and adenomatous polyposis coli (APC), reversing progrowth GH signals. p53 was also induced in skin fibroblasts derived from short-statured humans with mutant GHR. GH-deficient prophet of pituitary-specific positive transcription factor 1 (Prop1)−/− mice exhibited induced colon p53 levels, and cross-breeding them with Apcmin+/− mice that normally develop intestinal and colon tumors resulted in GH-deficient double mutants with markedly decreased tumor number and size. We also demonstrate that GH suppresses p53 and reduces apoptosis in human colon cell lines as well as in induced human pluripotent stem cell-derived intestinal organoids, and confirm in vivo that GH suppresses colon mucosal p53/p21. GH excess leads to decreased colon cell phosphatase and tensin homolog deleted on chromosome 10 (PTEN), increased cell survival with down-regulated APC, nuclear β-catenin accumulation, and increased epithelial–mesenchymal transition factors and colon cell motility. We propose that GH is a molecular component of the “field change” milieu permissive for neoplastic colon growth.

Total adiponectin, but not inflammatory markers C-reactive protein, tumor necrosis factor-α, interluekin-6 and monocyte chemoattractant protein-1, correlates with increasing glucose intolerance in pregnant Chinese-Americans

Elevated insulin, C‐reactive protein (CRP), tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, and monocyte chemoattractant protein (MCP)‐1 levels and decreased high molecular weight adiponectin (HMW‐APN) levels have been reported in Caucasians with gestational diabetes mellitus (GDM). No similar studies have been performed in Chinese women.

Total adiponectin, but not inflammatory markers C-reactive protein, tumor necrosis factor-α, interluekin-6 and monocyte chemoattractant protein-1, correlates with increasing glucose intolerance in pregnant Chinese–Americans (总脂联素，而不是炎症标志物包括C-反应蛋白、肿瘤坏死因子-α、白细胞介素-6以及单核细胞趋化蛋白-1与妊娠的华裔美国人的葡萄糖耐量受损加重有关)

Elevated insulin, C‐reactive protein (CRP), tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, and monocyte chemoattractant protein (MCP)‐1 levels and decreased high molecular weight adiponectin (HMW‐APN) levels have been reported in Caucasians with gestational diabetes mellitus (GDM). No similar studies have been performed in Chinese women.

Achieving a successful pregnancy in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a disease of complex and still poorly understood cause and of variable phenotypes. It is characterized by anovulation, hyperandrogenism, and polycystic ovaries. Infertility is commonly present. A variety of methods has been used successfully to achieve pregnancy in women with PCOS. Maintenance of pregnancy is complicated by a higher rate of premature spontaneous abortions and high risk of gestational diabetes, hypertension, and preeclampsia. However, with careful monitoring and treatment, the outcome of pregnancy in most women with PCOS is excellent.

E2F1-mediated human POMC expression in ectopic Cushing’s syndrome

Cushings syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushings syndrome). Hypercortisolemic features of ectopic Cushings syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushings syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushings cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushings syndrome.

Is there visceral adipose tissue (VAT) intracellular hypercortisolism in human obesity

The fact that obesity is a prominent feature of Cushings syndrome (systemic hypercortisolism of adrenocortical origin) stimulated a 40-year search for evidence of systemic hypercortisolism in human obesity. That search has failed to find such evidence. For the past 15 years, however, studies have been done to evaluate a possible alternative type of hypercortisolism in obesity, namely visceral adipose tissue (VAT) intracellular hypercortisolism. The current review summarizes the evidence published so far about this possibility. There have been three types of evidence studied: direct measurement of the VAT levels of 11β-hydroxysteroid dehydrogenase type I (11-HSD-1), which converts biologically inactive cortisone to biologically active cortisol; direct measurement of splanchnic cortisol production; and evaluation of the effect of a specific inhibitor of 11-HSD-1 on metabolic abnormalities associated with obesity, particularly diabetes mellitus. The results are complex and difficult to interpret. Our conclusion is that the presence of VAT intracellular hypercortisolism in human obesity is possible but unlikely.