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The transition from radioimmunoassay (RIA) to chemiluminescent enzyme immunoassay (CLEIA) for plasma aldosterone concentration (PAC) assays has raised concerns over its impact on primary aldosteronism (PA) diagnosis. This study investigated the correlation between PAC and renin values using RIA, CLEIA, and liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS), established cutoff values for PA diagnosis using the aldosterone-to-renin ratio (ARR) with PAC_CLEIA, and assessed the differences in PAC values by measuring weak mineralocorticoids (WMs). This retrospective study evaluated 312 serum PAC samples using RIA, CLEIA, and LC-MS/MS, and analyzed 315 plasma renin samples. Method correlations were assessed through Passing-Bablok regression. Receiver operating characteristic curves determined ARR cutoffs for PA diagnosis. WMs were quantified to evaluate their impact on ΔPAC (RIA-LC-MS/MS) through multiple regression analysis. PAC_CLEIA and PAC_LC-MS/MS values were highly correlated. ARRs derived from PAC_RIAs demonstrated more false positives and lower specificity than ARRs using PAC_CLEIA or PAC_LC-MS/MS. WMs significantly influenced ΔPAC in both the PA and non-PA groups. ARRs using PAC_CLEIA are valuable for determining PA cutoffs in clinical practice. The transition to PAC using CLEIA may enhance PA detection rates. WMs were found to interfere with PAC measurements in the RIA method, affecting outcomes., (© 2024. The Author(s).)

α-Synuclein (α-Syn) is a protein related to synucleinopathies with high expression in the central nervous system and erythrocytes which are a major source of peripheral α-Syn. Recent reports have suggested the presence of α-Syn within extracellular vesicles (EVs) derived from erythrocytes, potentially contributing to the pathogenesis of synucleinopathies. While Lewy bodies, intracellular inclusions containing aggregated α-Syn, are prominently observed within the brain, their occurrence in peripheral neurons implies the dissemination of synucleinopathy pathology throughout the body via the propagation of α-Syn. In this study, we found erythrocytes and circulating EVs obtained from plasma contained α-Syn, which was separated into four major forms using high-resolution clear native-PAGE and isoelectric focusing. Notably, erythrocyte α-Syn was classified into full-length and C-terminal truncated forms, with truncation observed between Y133 and Q134 as determined by LC-MS/MS analysis. Our finding revealed that C-terminally truncated α-Syn, which was previously reported to exist solely within the brain, was also present in erythrocytes and circulating EVs obtained from plasma., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemic lactic acidosis. Due to the rarity of FBPase deficiency, the mechanism by which the mutations cause enzyme activity loss still remains unclear. Here we identify compound heterozygous missense mutations of FBP1, c.491G>A (p.G164D) and c.581T>C (p.F194S), in an adult patient with hypoglycemic lactic acidosis. The G164D and F194S FBP1 mutants exhibit decreased FBP1 protein expression and a loss of FBPase enzyme activity. The biochemical phenotypes of all previously reported FBP1 missense mutations in addition to G164D and F194S are classified into three functional categories. Type 1 mutations are located at pivotal residues in enzyme activity motifs and have no effects on protein expression. Type 2 mutations structurally cluster around the substrate binding pocket and are associated with decreased protein expression due to protein misfolding. Type 3 mutations are likely nonpathogenic. These findings demonstrate a key role of protein misfolding in mediating the pathogenesis of FBPase deficiency, particularly for Type 2 mutations. This study provides important insights that certain patients with Type 2 mutations may respond to chaperone molecules., (© 2023. The Author(s).)

Although polymerase chain reaction (PCR) amplification and sequencing of the bacterial 16S rDNA region has numerous scientific applications, it does not provide DNA methylation information. Herein, we propose a simple extension for bisulfite sequencing to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical isolates or flora. Multiple displacement amplification without DNA denaturation was used to preferentially pre-amplify single-stranded bacterial DNA after bisulfite conversion. Following the pre-amplification, the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing, enabling the simultaneous identification of DNA methylation status and sequence data. We used this approach (termed sm16S rDNA PCR/sequencing) to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii and different methylation motifs among Enterococcus faecalis strains from small volumes of clinical specimens. Further, our analysis suggested that M. MmnI may be correlated to erythromycin resistance. Thus, sm16S rDNA PCR/sequencing is a useful extension method for analyzing the DNA methylation of 16S rDNA regions in a microflora, providing additional information not provided by conventional PCR. Given the relationship between DNA methylation status and drug resistance in bacteria, we believe this technique can be effectively applied in clinical sample testing., (© 2023. The Author(s).)

α-Synuclein is a protein linked to various synuclein-associated diseases ('synucleinopathies'), including Parkinson's disease, dementia with Lewy Bodies and multiple system atrophy, and is highly expressed in the central nervous system and in erythrocytes. Moreover, α-synuclein-containing erythrocyte-derived extracellular vesicles may be involved in the pathogenesis of synucleinopathies and their progression across the blood-brain barrier. Several post-translational modifications of α-synuclein have been reported in brain inclusions, including S129 phosphorylation, but fewer have been found in erythrocytes. In this study, we analysed the post-translational modifications of erythrocyte α-synuclein using liquid chromatography-mass spectrometry. We found that all lysine residues in the α-synuclein protein could be modified by acetylation, glycation, ubiquitination or SUMOylation but that phosphorylation, nitration and acylation were uncommon minor post-translational modifications in erythrocytes. Since the post-translational modification of lysine residues has been implicated in both membrane association and protein clearance, our findings provide new insight into how synucleinopathies may progress and suggest possible therapeutic strategies designed to target α-synuclein., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The clinical characteristics of growth hormone (GH)-producing pituitary adenomas/somatotroph pituitary neuroendocrine tumors (GHomas/somatotroph PitNETs) vary across patients. In this study, we aimed to integrate the genetic alterations, protein expression profiles, transcriptomes, and clinical characteristics of GHomas/somatotroph PitNETs to identify molecules associated with acromegaly characteristics. Targeted capture sequencing and copy number analysis of 36 genes and nontargeted proteomics analysis were performed on fresh-frozen samples from 121 sporadic GHomas/somatotroph PitNETs. Targeted capture sequencing revealed GNAS as the only driver gene, as previously reported. Classification by consensus clustering using both RNA sequencing and proteomics revealed many similarities between the proteome and the transcriptome. Gene ontology analysis was performed for differentially expressed proteins between wild-type and mutant GNAS samples identified by nontargeted proteomics and involved in G protein-coupled receptor (GPCR) pathways. The results suggested that GNAS mutations impact endocrinological features in acromegaly through GPCR pathway induction. ATP2A2 and ARID5B correlated with the GH change rate in the octreotide loading test, and WWC3, SERINC1, and ZFAND3 correlated with the tumor volume change rate after somatostatin analog treatment. These results identified a biological connection between GNAS mutations and the clinical and biochemical characteristics of acromegaly, revealing molecules associated with acromegaly that may affect medical treatment efficacy., (© 2022. The Author(s).)

Cushing's disease causes numerous metabolic disorders, cognitive decline, and sarcopenia, leading to deterioration of the general health in older individuals. Cushing's disease can be treated with transsphenoidal surgery, but thus far, surgery has often been avoided in older patients. We herein report an older woman with Cushing's disease whose cognitive impairment and sarcopenia improved after transsphenoidal surgery. Although cognitive impairment and sarcopenia in most older patients show resistance to treatment, our case indicates that normalization of the cortisol level by transsphenoidal surgery can be effective in improving the cognitive impairment and muscle mass loss caused by Cushing's disease.

The microRNA (miR) miR-874, a potential tumour suppressor, causes cell death via target gene suppression in various cancer types. Mevalonate pathway inhibition also causes cell death in breast cancer. However, the relationship between the mevalonate pathway and miR-874-induced apoptosis or its association with the tumour suppressor p53 has not been elucidated. We identified phosphomevalonate kinase (PMVK), a key mevalonate pathway enzyme, and sterol regulatory element-binding factor 2 (SREBF2), the master cholesterol biosynthesis regulator, as direct miR‑874 targets. Next-generation sequencing analysis revealed a significant miR-874-mediated downregulation of PMVK and SREBF2 gene expression and p53 pathway enrichment. Luciferase reporter assays showed that miR-874 directly regulated PMVK and SREBF2. miR-874-induced apoptosis was p53 dependent, and single-cell RNA sequencing analysis demonstrated that miR-874 transfection resulted in apoptosis and p53 pathway activation. Downregulation of PMVK expression also caused cell cycle arrest and p53 pathway activation, which was rescued by geranylgeranyl pyrophosphate (GGPP) supplementation. Analysis of The Cancer Genome Atlas (TCGA) database indicated a negative correlation between miR-874 and PMVK expression and between miR-874 and SREBF2 expression. These findings suggest that miR-874 suppresses the mevalonate pathway by targeting SREBF2 and PMVK, resulting in GGPP depletion, which activates the p53 pathway and promotes cycle arrest or apoptosis., (© 2022. The Author(s).)

The liver stores glycogen and releases glucose into the blood upon increased energy demand. Group 2 innate lymphoid cells (ILC2) in adipose and pancreatic tissues are known for their involvement in glucose homeostasis, but the metabolic contribution of liver ILC2s has not been studied in detail. Here we show that liver ILC2s are directly involved in the regulation of blood glucose levels. Mechanistically, interleukin (IL)-33 treatment induces IL-13 production in liver ILC2s, while directly suppressing gluconeogenesis in a specific Hnf4a/G6pc-high primary hepatocyte cluster via Stat3. These hepatocytes significantly interact with liver ILC2s via IL-13/IL-13 receptor signaling. The results of transcriptional complex analysis and GATA3-ChIP-seq, ATAC-seq, and scRNA-seq trajectory analyses establish a positive regulatory role for the transcription factor GATA3 in IL-13 production by liver ILC2s, while AP-1 family members are shown to suppress IL-13 release. Thus, we identify a regulatory role and molecular mechanism by which liver ILC2s contribute to glucose homeostasis., (© 2022. The Author(s).)

Ceiling culture-derived preadipocytes (ccdPAs) and adipose-derived stem cells (ASCs) can be harvested from human subcutaneous fat tissue using the specific gravity method. Both cell types possess a similar spindle shape without lipid droplets. We previously reported that ccdPAs have a higher adipogenic potential than ASCs, even after a 7-wk culture. We performed a genome-wide epigenetic analysis to examine the mechanisms contributing to the adipogenic potential differences between ccdPAs and ASCs. Methylation analysis of cytosines followed by guanine (CpG) using a 450-K BeadChip was performed on human ccdPAs and ASCs isolated from three metabolically healthy females. Chromatin immunoprecipitation sequencing was performed to evaluate trimethylation at lysine 4 of histone 3 (H3K4me3). Unsupervised machine learning using t-distributed stochastic neighbor embedding to interpret 450,000-dimensional methylation assay data showed that the cells were divided into ASC and ccdPA groups. In Kyoto Encyclopedia of Genes and Genomes pathway analysis of 1,543 genes with differential promoter CpG methylation, the peroxisome proliferator-activated receptor (PPAR) and adipocytokine signaling pathways ranked in the top 10 pathways. In the PPARγ gene, H3K4me3 peak levels were higher in ccdPAs than in ASCs, whereas promoter CpG methylation levels were significantly lower in ccdPAs than in ASCs. Similar differences in promoter CpG methylation were also seen in the fatty acid-binding protein 4 and leptin genes. In conclusion, we analyzed the epigenetic status of adipogenesis-related genes as a potential mechanism underlying the differences in adipogenic differentiation capability between ASCs and ccdPAs.

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The incidence of thyroid carcinoma has been increasing worldwide. This is interpreted as an increase in the incidental detection of papillary thyroid microcarcinomas (PTMCs). However, mortality has not changed, suggesting overdiagnosis and overtreatment. Prospective clinical trials of active surveillance for low-risk PTMC (T1aN0M0) have been conducted in two Japanese institutions since the 1990s. Based on the favorable outcomes of these trials, active surveillance has been gradually adopted worldwide. A task force on the management of PTMC in adults organized by the Japan Thyroid Association therefore conducted a systematic review and has produced the present position paper based on the scientific evidence concerning active surveillance. This paper indicates evidence for the increased incidence of PTMC, favorable surgical outcomes for low-risk PTMC, recommended criteria for diagnosis using fine needle aspiration cytology, and evaluation of lymph node metastasis (LNM), extrathyroidal extension (ETE) and distant metastasis. Active surveillance has also been reported with a low incidence of disease progression and no subsequent recurrence or adverse events on survival if conversion surgery was performed at a slightly advanced stage. Active surveillance is a safe and valid strategy for PTMC, because it might preserve physical quality of life and reduce 10-year medical costs. However, some points should be noted when performing active surveillance. Immediate surgery is needed for PTMC showing high-risk features, such as clinical LNM, ETE or distant metastasis. Active surveillance should be performed under an appropriate medical team and should be continued for life.

Rathke's cleft cyst (RCC) is a common incidental tumor in the hypothalamic-pituitary region. Some reports have shown that the clinical symptoms and endocrine functions of symptomatic RCCs are temporarily improved by glucocorticoid administration. However, it is still unknown whether glucocorticoid treatment is effective for symptomatic RCCs according to long-term observations. In this study, we describe the long-term clinical outcomes of two cases of glucocorticoid-treated biopsy-proven secondary hypophysitis caused by RCCs. We summarize the symptoms, imaging findings, and endocrine evaluations of two symptomatic RCC patients with concomitant hypophysitis before and after prednisolone treatment. In both evaluated cases, visual impairments and altered endocrine parameters were present due to chiasm and stalk compression; these outcomes improved after shrinkage of RCCs in response to prednisolone administration, and partial recovery of anterior pituitary hormone secretion was observed. However, in both cases, the deficits in anterior pituitary hormone secretion recurred, possibly due to persistent inflammatory infiltration in the RCCs and pituitary glands. After relapse of hypophysitis, anterior hormone secretion did not fully recover. In our cases of secondary hypophysitis caused by RCCs, prednisolone administration had an early effect of cyst shrinkage, followed by partial improvements in clinical symptoms and pituitary functions. However, long-term observation showed that prednisolone treatment did not contribute to complete improvement in anterior pituitary hormone dysfunction.

Objectives: To investigate whether the result of the 1-mg dexamethasone suppression test can predict the improvement of comorbidities after adrenalectomy in patients with subclinical Cushing syndrome., Methods: This retrospective study included 117 subclinical Cushing syndrome patients who underwent adrenalectomy. The numbers of prescribed drugs for metabolic comorbidities and the clinical variables at diagnosis were compared with those at the follow up. Patients were classified into subgroups according to the result of the 1-mg dexamethasone suppression test., Results: Significant improvements in blood pressure, serum cholesterol and body mass index were observed. Furthermore, a significant improvement in glycated hemoglobin was observed in patients with diabetes mellitus. These improvements led to a discontinuation or reduction of prescribed drugs after surgery. In addition, the greatest reduction of prescribed drugs was observed in patients whose serum cortisol levels were between 1.8 and 3.0 µg/dL after the 1-mg dexamethasone suppression test., Conclusions: The result of the 1-mg dexamethasone suppression test can be a useful factor predicting the improvement of comorbidities after adrenalectomy. Current data might give us a new insight into the decision-making for the treatment of subclinical Cushing syndrome., (© 2020 The Japanese Urological Association.)

TAS4464, a potent, selective small molecule NEDD8-activating enzyme (NAE) inhibitor, leads to inactivation of cullin-RING E3 ubiquitin ligases (CRLs) and consequent accumulations of its substrate proteins. Here, we investigated the antitumor properties and action mechanism of TAS4464 in acute myeloid leukemia (AML). TAS4464 induced apoptotic cell death in various AML cell lines. TAS4464 treatments resulted in the activation of both the caspase-9-mediated intrinsic apoptotic pathway and caspase-8-mediated extrinsic apoptotic pathway in AML cells; combined treatment with inhibitors of these caspases markedly diminished TAS4464-induced apoptosis. In each apoptotic pathway, TAS4464 induced the mRNA transcription of the intrinsic proapoptotic factor NOXA and decreased that of the extrinsic antiapoptotic factor c-FLIP. RNA-sequencing analysis showed that the signaling pathway of the CRL substrate c-Myc was enriched after TAS4464 treatment. Chromatin immunoprecipitation (ChIP) assay revealed that TAS4464-induced c-Myc bound to the PMAIP1 (encoding NOXA) and CFLAR (encoding c-FLIP) promoter regions, and siRNA-mediated c-Myc knockdown neutralized both TAS4464-mediated NOXA induction and c-FLIP downregulation. TAS4464 activated both caspase-8 and caspase-9 along with an increase in NOXA and a decrease in c-FLIP, resulting in complete tumor remission in a human AML xenograft model. These findings suggest that NAE inhibition leads to anti-AML activity via a novel c-Myc-dependent apoptosis induction mechanism.

Objective: To assess the correlation value between adrenal venous sampling (AVS) and I-6β-iodomethyl-19-norcholesterol (NP-59) adrenal scintigraphy in differentiating aldosterone-producing adenoma (APA) from bilateral idiopathic hyperaldosteronism (BHA), and the use of NP-59 scintigraphy as an alternative to AVS., Methods: Overall, 29 patients with APA or BHA who underwent AVS and dexamethasone-suppression NP-59 scintigraphy were included between 2010 and 2017. The correlation value between AVS and dexamethasone-suppression NP-59 scintigraphy was assessed using each lateralisation index (LIAVS and LI1NP-59). Tumour presence and size were evaluated using computed tomography. The sensitivity and specificity of dexamethasone-suppression NP-59 scintigraphy for APA according to each lateralisation index threshold were calculated., Results: Of 29 patients, 12 presented with APA and 17 with BHA according to AVS. The correlation value between LIAVS and LI1NP-59 was 0.63 (P < 0.001). If the cut-off points were 2.55 and 1.80 in all cases, the sensitivity and specificity were 0.33 and 1.00 as well as 0.58 and 0.94, respectively. In adrenal microtumours (maximum diameter ≤10 mm), no cases revealed a cut-off point of >1.8. However, in adrenal macrotumours (maximum diameter >10 mm), the cut-off point of 2.55 represented the best compromise (sensitivity: 0.44; specificity: 1.00)., Conclusion: NP-59 scintigraphy can be used as an alternative to AVS if there is a strong lateralisation on NP-59 scintigraphy and adrenal macrotumours observed on the computed tomography when AVS is technically challenging, particularly in the right adrenal vein cannulation, and if contraindications, such as allergy to contrast materials and renal failure, are observed.

Background: Pressure sores are sometimes refractory to treatment, often due to malnutrition. Small intestinal bacterial overgrowth (SIBO) obstructs absorption in the digestive tract and causes malnutrition. However, little is known about the association between pressure sore wound healing and SIBO. Here, we report a case of a patient with a refractory sacral pressure sore and SIBO., Case Presentation: A 66-year-old woman who was spinal cord injured 14 years before visiting our hospital presented with the chief complaint of a sacral pressure sore, 10.0 × 6.5 cm in size, which was refractory to treatment. Physical examination showed abdominal distension and emaciation, with a body mass index of 15. Further examination revealed elevated serum alkaline phosphatase (1260 U/L), bilateral tibial fracture, multiple rib fracture, and osteoporosis. We diagnosed the patient with osteomalacia with vitamin D deficiency. Despite oral supplementation, serum levels of calcium, phosphorous, and vitamin D remained low. Also, despite concentrative wound therapy for the sacral pressure sore by plastic surgeons, no wound healing was achieved. Due to a suspicion of disturbances in nutrient absorption, we performed bacterial examination of collected gastric and duodenal fluid, which showed high numbers of bacteria in gastric content (10 4 E. coli, 10 5 Streptococcus species, and 10 5 Neisseria species) and duodenal content (10 6 E. coli, 10 4 Candida glabrata). Therefore, we diagnosed the patient with SIBO and started selective decontamination of the digestive tract using polymyxin B sulfate and amphotericin B. After starting treatment for SIBO, the sacral pressure sore began to heal and was nearly healed after 285 days. The patient's serum levels of calcium, phosphorous, vitamin D, and other fat-soluble vitamins also gradually increased after starting treatment for SIBO., Conclusion: We report a case of a patient with a refractory sacral pressure sore that healed after starting treatment for SIBO. We conclude that SIBO may be an overlooked cause of malnutrition and poor wound healing in patients with chronic pressure sores.

Objective: Accurate assessment and localization of aldosterone-producing adenomas (APAs) are essential for the treatment of primary aldosteronism (PA). Although adrenal venous sampling (AVS) is the standard method of reference for subtype diagnosis in PA, controversy exists concerning the criteria for its interpretation. This study aims to determine better indicators that can reliably predict subtypes of PA., Method: Retrospective, single-cohort analysis including 209 patients with PA who were subjected to AVS. Eighty-two patients whose plasma aldosterone concentrations (PAC) were normalized after surgery were histopathologically or genetically diagnosed with APA. The accuracy of image findings was compared to AVS results. Receiver operating characteristic (ROC) curve analysis between the operated and the no-apparent laterality groups was performed using AVS parameters and loading test for diagnosis of PA., Result: Agreement between image findings and AVS results was 56.3%. ROC curve analysis revealed that the lateralization index (LI) after adrenocorticotropin stimulation cutoff was 2.40, with 98.8% sensitivity and 97.1% specificity. The contralateral suppression index (CSI) cutoff value was 1.19, with 98.0% sensitivity and 93.9% specificity. All patients over the LI and CSI cutoff values exhibited unilateral subtypes. Among the loading test, the best classification accuracy was achieved using the PAC reduction rate after a saline infusion test (SIT) >33.8%, which yielded 87.2% sensitivity or a PAC after a SIT <87.9 pg/mL with 86.2% specificity for predicting bilateral PA., Conclusion: The combined criteria of the PAC reduction rate and PAC after the SIT can determine which subset of patients with APA who should be performed AVS for validation., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Hashimoto's thyroiditis is the most common cause of hypothyroidism. Patients usually respond well to oral synthetic thyroxine (levothyroxine); however, for unknown reasons some individuals present with treatment-resistant Hashimoto thyroiditis. In cases of cancer and certain infectious diseases, the ATP binding cassette (ABC) transporters have been implicated in multidrug resistance, and we hypothesized and investigated a role of ABC transporters in drug-resistant Hashimoto's thyroiditis., Case Description: The patient whose case we report had a history of Hashimoto's thyroiditis, immune thrombocytopenia, and refractory hypertension, with varying treatment resistance to the oral medications prescribed for each condition. In order to establish or exclude a genetic basis for her illness, we examined the patient's gene expression profiles using peripheral blood leukocytes, and found that ABCG2/BCRPexpression was significantly high compared with healthy volunteers. Also, the increased daunomycin efflux capacity of our patient's lymphocytes was successfully inhibited by fumitremorgin C, a specific ABCG2/BCRP inhibitor, and the patient's level of thyroid-stimulating hormone increased by 248.6% after administration of intact levothyroxine tablets but decreased by 45.1% when tablets were crushed. Her average blood pressure decreased from 166.3/108.5 mmHg to 125.9/78.8 mmHg when switching from intact to crushed losartan tablets., Conclusions: High expression and accelerated efflux transporter activity of ABCG2/BCRP in the small intestine are expected to contribute to the ineffectiveness of orally administered intact tablets in cases with treatment-resistant Hashimoto's thyroiditis, and crushed tablets can be more effective for some of these patients., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Adipose-derived stem cells and ceiling culture-derived preadipocytes can be harvested from subcutaneous adipose tissue. Little is known about the epigenetic differences, which may contribute to differences in osteogenic potential, between these cell types. The purpose of this study was to address the osteogenic potential and underlying epigenetic status of adipose-derived stem cells and ceiling culture-derived preadipocytes., Methods: Adipose-derived stem cells and ceiling culture-derived preadipocytes were cultured from abdominal subcutaneous fat tissues of four metabolically healthy, lean female patients. After 7 weeks of culture, cellular responses to osteogenic differentiation media were examined. To evaluate the osteogenic potentials of undifferentiated adipose-derived stem cells and ceiling culture-derived preadipocytes, two types of epigenetic assessment were performed using next-generation sequencing: DNA methylation assays with the Human Methylation 450K BeadChip, and chromatin immunoprecipitation assays for trimethylation of histone H3 at lysine 4., Results: Human ceiling culture-derived preadipocytes showed greater osteogenic differentiation ability than did adipose-derived stem cells. In an epigenetic survey of the promoters of four osteogenic regulator genes (RUNX2, SP7, ATF4, and BGLAP), the authors found a general trend toward decreased CpG methylation and increased trimethylation of histone H3 at lysine 4 levels in ceiling culture-derived preadipocytes as compared to adipose-derived stem cells, indicating that these genes were more likely to be highly expressed in ceiling culture-derived preadipocytes., Conclusions: The surveyed epigenetic differences between adipose-derived stem cells and ceiling culture-derived preadipocytes were consistent with the observed differences in osteogenic potential. These results enhance the authors' understanding of these cells and will facilitate their further application in regenerative medicine.

The tumor suppressor gene p53 is mutated in approximately more than 50% of human cancers. p53 is also referred to as the "cellular gatekeeper" or "guardian of the genome" because it protects the body from spreading mutated genome induced by various stress. When the cells receives stimuli such as DNA damage, oncogene activation, oxidative stress or undernutrition, p53 gives rise to a number of cellular responses, including cell cycle arrest, apoptosis, cellular senescence and metabolic adaptation. Related to energy metabolisms, it has been reported that p53 reduces glycolysis and enhances mitochondrial respiration. p53 is also involved in the regulation of other cellular metabolism and energy production systems: amino acid metabolism, fatty acid metabolism, nucleic acid metabolism, anti-oxidation, mitochondrial quality control, and autophagy. Moreover, recent studies have shown that p53 gene polymorphisms affect life expectancy and lifestyle-related disease such as type 2 diabetes, suggesting that there is a certain relationship between p53 function and metabolic disorders. In addition, mutant p53 protein does not only lose the tumor suppressor function, but it also gains novel oncogenic function and contributes to tumor development, involving cellular metabolism modification. Therefore, the importance of multifunctionality of p53, particularly with regard to intracellular metabolisms, arouses therapeutic interest and calls attention as the key molecule among cancer, lifestyle-related diseases and life expectancy.

We herein report a 75-year-old woman with insulin-treated diabetes and metastatic anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer who received ceritinib, a second-generation ALK inhibitor, and achieved dramatic tumor reduction. However, her fasting blood glucose increased, particularly markedly in the first two weeks after ceritinib administration, and did not normalize even increasing the total insulin dose. After discontinuing ceritinib, her glucose levels rapidly reduced. Ceritinib can aggravate hyperglycemia in patients with diabetes who lack compensatory insulin secretion, due to its inhibitory effects on the insulin receptor. Careful monitoring for ceritinib-induced hyperglycemia should be performed, especially in the first two weeks after ceritinib administration.

The tumor suppressor p53 regulates multiple cellular functions, including energy metabolism. Metabolic deregulation is implicated in the pathogenesis of some cancers and in metabolic disorders and may result from the inactivation of p53 functions. Using RNA sequencing and ChIP sequencing of cancer cells and preadipocytes, we demonstrate that p53 modulates several metabolic processes via the transactivation of energy metabolism genes including dihydropyrimidinase-like 4 ( DPYSL4 ). DPYSL4 is a member of the collapsin response mediator protein family, which is involved in cancer invasion and progression. Intriguingly, DPYSL4 overexpression in cancer cells and preadipocytes up-regulated ATP production and oxygen consumption, while DPYSL4 knockdown using siRNA or CRISPR/Cas9 down-regulated energy production. Furthermore, DPYSL4 was associated with mitochondrial supercomplexes, and deletion of its dihydropyrimidinase-like domain abolished its association and its ability to stimulate ATP production and suppress the cancer cell invasion. Mouse-xenograft and lung-metastasis models indicated that DPYSL4 expression compromised tumor growth and metastasis in vivo. Consistently, database analyses demonstrated that low DPYSL4 expression was significantly associated with poor survival of breast and ovarian cancers in accordance with its reduced expression in certain types of cancer tissues. Moreover, immunohistochemical analysis using the adipose tissue of obese patients revealed that DPYSL4 expression was positively correlated with INFg and body mass index in accordance with p53 activation. Together, these results suggest that DPYSL4 plays a key role in the tumor-suppressor function of p53 by regulating oxidative phosphorylation and the cellular energy supply via its association with mitochondrial supercomplexes, possibly linking to the pathophysiology of both cancer and obesity., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Context: Necrolytic migratory erythema (NME) occurs in approximately 70% of patients with glucagonoma syndrome. Excessive stimulation of metabolic pathways by hyperglucagonemia, which leads to hypoaminoacidemia, contributes to NME pathogenesis. However, the molecular pathogenesis of glucagonoma and relationships between metabolic abnormalities and clinical symptoms remain unclear., Patient: A 53-year-old woman was referred to our hospital with a generalized rash and weight loss. NME was diagnosed by histopathological examination of skin biopsy tissue. Laboratory tests revealed diabetes, hyperglucagonemia, marked insulin resistance, severe hypoaminoacidemia, ketosis, and anemia. Enhanced computed tomography scans detected a 29-mm pancreatic hypervascular tumor, which was eventually diagnosed as glucagonoma. Preoperative treatment with octreotide long-acting release reduced the glucagon level, improved the amino acid profile, and produced NME remission. Surgical tumor excision normalized the metabolic status and led to remission of symptoms, including NME., Interventions: Whole-exome sequencing (WES) and subsequent targeted capture sequencing, followed by Sanger sequencing and pyrosequencing, identified biallelic alteration of death-domain associated protein (DAXX) with a combination of loss of heterozygosity and frameshift mutations (c.553&#95;554del:p.R185fs and c.1884dupC:p.C629fs) in the glucagonoma. Consistently, immunohistochemistry confirmed near-absence of DAXX staining in the tumor cells. Tumor expression of glucagon and somatostatin receptor subtype 2 and 3 messenger RNA was markedly upregulated., Conclusions: This is a report of glucagonoma with biallelic inactivation of DAXX determined by WES. The tumor manifested as glucagonoma syndrome with generalized NME. This case showed the relationship between hypoaminoacidemia and NME status. Further investigations are required to elucidate the underlying mechanisms of NME onset and glucagonoma tumorigenesis.

Objectives: To identify pre-treatment factors affecting the duration of post-surgical steroid replacement in patients undergoing adrenalectomy for subclinical Cushing syndrome., Methods: The present retrospective analysis included 64 patients who underwent unilateral laparoscopic adrenalectomy for subclinical Cushing syndrome. Adrenal tumor and contralateral adrenal sizes together with various clinical factors were studied in association with the duration of post-surgical steroid replacement. Adrenal tumor and contralateral adrenal size were measured at the level of the maximum transverse plane of the adrenal glands using computed tomography scan or magnetic resonance imaging. Cox's proportional hazards model was used for the statistical analysis., Results: All 64 patients were treated with post-surgical steroid replacement after adrenalectomy. The median duration of the steroid treatment was 6 months. When assessing the duration of post-surgical steroid replacement, contralateral adrenal volume <0.745 cm 3 , contralateral adrenal width <6.15 mm and serum cortisol after a 1-mg dexamethasone suppression test >2.65 μg/dL were significant predictors of prolonged post-surgical steroid treatment on univariate analysis. On multivariate analysis, contralateral adrenal width <6.15 mm was the only independent predictive factor for the prolonged post-surgical steroid replacement., Conclusions: Contralateral adrenal width seems to represent a significant predictive factor for the duration of post-surgical steroid replacement in subclinical Cushing syndrome patients. Pre-surgical assessment of image findings might help clinicians determine the total duration of steroid therapy after adrenalectomy., (© 2018 The Japanese Urological Association.)

We herein report the case of a 25-year-old woman who presented with severe headache and visual field defects after childbirth. Magnetic resonance imaging revealed marked swelling of the pituitary gland, and an endocrinological examination revealed panhypopituitarism and diabetes insipidus. An immunohistological analysis of a transsphenoidal biopsy sample of the pituitary gland showed the significant accumulation of an immunogloblin G4 (IgG4)-positive population, leading to the diagnosis of IgG4-related hypophysitis. The patient was treated with prednisolone, which markedly reduced the swelling of the pituitary gland, in association with recovery of the pituitary function. This is a rare case of biopsy-proven IgG4-related hypophysitis with a postpartum onset.

Objective: Familial dysalbuminemic hyperthyroxinemia (FDH) is caused by abnormal human serum albumin (HSA) with an increased thyroxine (T4) affinity leading to euthyroid hyperthyroxinemia. One- and 2-step immunoassays of serum samples from FDH patients (e.g., Japanese patients) with the HSA R218P mutation can yield false-positive free thyroxine (FT4) results. Therefore, it is difficult to distinguish FDH from syndrome of inappropriate secretion of thyroid-stimulating hormone (TSH) (e.g., syndrome of resistance to thyroid hormone, TSH-producing pituitary adenoma), even when multiple assays are used. To investigate T4 to HSA binding, we examined serum samples from 7 patients from 3 Japanese families with FDH. Clinically, abnormal thyroid function tests were noted in pregnant Patient 1. Patients 2 and 3 had histories of inappropriate treatment with antithyroid drugs and surgery., Methods: All patients and affected family members were diagnosed with FDH using direct sequencing analysis. Gel filtration high-performance liquid chromatography was used for the biochemical analyses., Results: The genomic analysis revealed a heterozygous missense mutation in HSA (R218P). In FDH patient sera, the albumin effluent corresponded to the peaks for total T4 (TT4); approximately 60% of the T4 in the effluent was detected as FT4. The results for the albumin effluent from healthy volunteer and TSHoma patient sera showed no corresponding TT4 peak., Conclusion: In the FDH patients, a relatively larger quantity of T4 was bound to abnormal HSA. This bound T4 was measured as FT4 during the analysis., Abbreviations: F = free; FDH = familial dysalbuminemic hyperthyroxinemia; HPLC = high-performance liquid chromatography; HSA = human serum albumin; PCR = polymerase chain reaction; SITSH = syndrome of inappropriate secretion of TSH; T = total; T3 = triiodothyronine; T4 = thyroxine; TSH = thyroid-stimulating hormone; WT = wild-type.

Background: A functional pituitary adenoma can produce multiple anterior-pituitary hormones, such as growth hormone (GH) -producing adenomas (GHoma) with prolactin or thyrotropin stimulating hormone production in the same lineage. However, it is very rare that acromegaly shows subclinical Cushing's disease (SCD) beyond the lineage. Here we describe the involvement of intratumoral coexistence with 2 types of hormone-producing cells associated with different lineage in acromegaly concomitant with SCD., Case Presentation: In our study, we performed clinical evaluation of the patient showing acromegaly with SCD. To elucidate the mechanisms of this pathology, we analyzed immunohistochemistry and gene expression of anterior-pituitary hormones and transcriptional factors in the resected pituitary tumor. On immunohistochemical staining, most of the tumor cells were strongly stained for GH antibody, while some cells were strongly positive for adrenocorticotropic hormone (ACTH). Gene expression analysis of a transsphenoidal surgery sample of the pituitary gland revealed that ACTH-related genes, such as POMC, Tpit, and NeuroD1 mRNA, had higher expression in the tumor tissue than the nonfunctional adenoma but lower expression compared to an adenoma of typical Cushing's disease. Further, double-labeling detection methods with a fluorescent stain for ACTH and GH demonstrated the coexistence of ACTH-positive cells (GH-negative) among the GH-positive cells in the tumor. Additionally, Pit-1 expression was reduced in the ACTH-positive cells from tumor tissue primary culture., Conclusion: Here we described a case of a pituitary tumor diagnosed with acromegaly associated with SCD. We performed quantitative-expression analyses of transcriptional factors of the tumor tissue and immunohistochemistry analysis of tumor-derived primary culture cells, which suggested that the multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells caused acromegaly associated with SCD.

Background: Laparoscopic adrenalectomy has been established as a standard surgical method for unilateral primary aldosteronism. Meanwhile, the background characteristics of the patients undergoing adrenalectomy have changed over the last 20 years. The aim of this study was to investigate the changes in hypertension cure rates after laparoscopic adrenalectomy during the last two decades., Methods: This retrospective clinical study included 176 patients who underwent unilateral laparoscopic adrenalectomy for primary aldosteronism from 1995 to 2015. The patients were divided into two groups by decade. The patients' baseline characteristics and the hypertension cure rates were compared between the two groups. Additionally, the values were re-examined based on predictive model predicting postoperative hypertension cure., Results: The hypertension cure rate decreased significantly from 51.8 to 31.1%. The following variables were significantly different between the two groups: age, sex, body mass index, history of diabetes mellitus, preoperative systolic and diastolic blood pressures, potassium level, and plasma renin activity., Conclusions: This study showed that the number of patients with unfavorable conditions for hypertension cure after adrenalectomy has recently increased. The treatment goal for primary aldosteronism is not only to cure the hypertension but also to prevent organ disorders due to inappropriate aldosterone levels. Therefore, we recommend laparoscopic adrenalectomy for unilateral primary aldosteronism, even if hypertension is not always cured postoperatively. However, clinicians need to fully explain the postoperative hypertension outcomes to primary aldosteronism patients.

Context: Pheochromocytoma is a catecholamine-producing tumor that originates from adrenal chromaffin cells and is capable of secreting various hormones, including ACTH., Case Description: A 56-year-old female presented with Cushingoid appearance and diabetic ketoacidosis. Endocrinological examinations demonstrated ectopic ACTH production with hypercortisolemia and excess urinary cortisol accompanied by elevated plasma and urine catecholamines. Computed tomography revealed a large left adrenal tumor with bilateral adrenal enlargement. Metaiodobenzylguanidine scintigraphy revealed abnormal accumulation in the tumor, which was eventually diagnosed as pheochromocytoma with ectopic ACTH secretion with subsequent manifestation of Cushing's syndrome. Ectopic ACTH secretion and catecholamine production were blocked by metyrapone treatment, whereas dexamethasone paradoxically increased ACTH secretion. Left adrenalectomy resulted in complete remission of Cushing's syndrome and pheochromocytoma., In Vitro Studies: Immunohistological analysis revealed that the tumor contained two functionally distinct chromaffin-like cell types. The majority of tumor cells stained positive for tyrosine hydroxylase (TH), whereas a minor population of ACTH-positive tumor cells was negative for TH. Furthermore, gene expression and in vitro functional analyses using primary tumor tissue cultures demonstrated that dexamethasone facilitated ACTH as well as catecholamine secretion with parallel induction of proopiomelanocortin (POMC), TH, and phenylethanolamine N-methyltransferase mRNA, supporting a glucocorticoid-dependent positive-feedback loop of ACTH secretion in vivo. DNA methylation analysis revealed that the POMC promoter of this tumor, particularly the E2F binding site, was hypomethylated., Conclusion: We present a case of ectopic ACTH syndrome associated with pheochromocytoma. ACTH up-regulation with paradoxical response to glucocorticoid, possibly through the hypomethylation of the POMC promoter, exacerbated the patient's condition.

Background: Many patients with primary aldosteronism (PA) show a significant decline in kidney function after adrenalectomy. Thus, PA patients who undergo surgery are at greater risk of both postoperative renal damage and new-onset metabolic events associated with renal insufficiency. The aim of this study was to explore postoperative changes in serum lipid levels and to identify risk factors associated with postoperative new-onset dyslipidemia in PA patients., Methods: The records of 57 Japanese patients who underwent unilateral laparoscopic adrenalectomy for PA were retrospectively surveyed. Clinical and biochemical data were evaluated at baseline and 12 months after surgery. Preoperative and postoperative estimated glomerular filtration (eGFR) and serum lipid profile, including triglycerides, high-density lipoprotein (HDL)-cholesterol and low-density lipoprotein (LDL)-cholesterol levels, were compared. Furthermore, uni- and multivariate analyses were performed to determine the predictors for postoperative new-onset dyslipidemia., Results: A significant decrease in eGFR and deterioration of serum lipid levels was identified postoperatively in most patients. Of the 39 patients without pre-existing dyslipidemia, 18 developed new-onset dyslipidemia postoperatively. Multivariate analysis identified preoperative lower eGFR and higher body mass index as independent predictors for new-onset dyslipidemia after surgery. On univariate analyses, additional factors associated with new-onset dyslipidemia included older age, male sex, higher LDL-cholesterol, and higher LDL/HDL ratio., Conclusions: PA patients had a higher risk of postoperative new-onset or progressive dyslipidemia. Clinicians should pay attention to not only follow-up of renal impairment but also total management of new-onset metabolic events associated with renal insufficiency in PA patients.

Objective: Primary macronodular adrenal hyperplasia (PMAH) is considered a predominantly sporadic disease, but familial forms are well recognized. Genetic studies revealed germline mutations in the armadillo repeat containing 5 gene (ARMC5) in the majority of PMAH cases. Furthermore, somatic ARMC5 mutations, as different types of second-hit mutations and loss of heterozygosity have been reported in each adrenal nodule in PMAH. Here, we describe the involvement of ARMC5 alteration in a familial case of PMAH., Methods: In our study, we performed clinical and genetic evaluations in a mother and her son with familial PMAH. To search for mutations and deletion of ARMC5, we used Sanger sequencing and droplet digital polymerase chain reaction (ddPCR), respectively., Results: Both patients showed the same phenotype of subclinical Cushing syndrome, with mild excess of mineralocorticoids and vasopressin-responsive cortisol secretion. The ddPCR analysis demonstrated that both mother and son had germline deletions in exons 1 to 5 of the ARMC5 gene locus. Furthermore, Sanger sequencing of DNA from the right and left adrenal nodules as well as peripheral blood of the son revealed the presence of another germline, missense mutation in ARMC5 exon 3 (p.P347S)., Conclusion: This is the first report demonstrating germline deletion of ARMC5 in familial PMAH. In addition to investigating mutations, germline and somatic deletions of ARMC5 could be examined by ddPCR, which permits rapid and accurate evaluation of the ARMC5 allelic status.

Background: The number of antihypertensive drug classes cannot accurately reflect the total consumption of antihypertensive drugs used to control blood pressure. The defined daily dose has been adopted to permit consumption analysis of many prescribed drugs. The aim of the present study was to assess postoperative changes in antihypertensive drug consumption in patients with primary aldosteronism using the defined daily dose as the unit of measurement., Methods: This retrospective study included 110 Japanese patients who underwent unilateral laparoscopic adrenalectomy between 1995 and 2012. Antihypertensive drug doses were calculated according to the standard of the defined daily dose recommended by the World Health Organization to compare drug use. After assessing postoperative changes in antihypertensive drug consumption, univariate and multivariate analyses were performed to identify clinical predictors for a 75% or greater decrease in the defined daily dose., Results: Consumption of antihypertensive drugs decreased postoperatively in 95.4% of patients. The median decrease in the defined daily dose was 76.8%. A postoperative decrease of 75% or greater in the defined daily dose was confirmed in 52.7% of patients. Multivariate analysis identified no medical history of cardiovascular disease, low body mass index, and short duration of hypertension as independent predictors of a postoperative decrease of 75% or greater in the defined daily dose., Conclusion: The defined daily dose is a useful tool for assessing total changes in the consumption of antihypertensive drugs in patients with primary aldosteronism. Using the defined daily dose, clinicians could explain in detail to patients with primary aldosteronism the predicted postoperative change in antihypertensive drug consumption., (Copyright © 2014. Published by Elsevier B.V.)

Purpose: Adrenal vein (AV) sampling (AVS) is the diagnostic gold standard for primary aldosteronism (PA), but right-sided AVS is difficult. We compared detection of AVs by selective retrograde CT adrenal venography (SRCTAV) with digital subtraction angiography (DSA)., Materials and Methods: Data on 29 subjects (11 males, mean age 55 y) with increased serum aldosterone concentrations (SAC) and a diagnosed right or left aldosterone-producing tumor (APT) by AVS who underwent laparoscopic adrenalectomy were retrospectively analyzed. Before AVS, visualizing AVs was attempted by DSA and SRCTAV (Aquilion). If after the adrenocorticotropic hormone loading test serum cortisol concentration (SCC) from either AV was >200 μg/dl, AVS was considered successful. If the SAC/SCC ratio for one side was ≥4 times higher than the other side, we diagnosed a one-sided APT., Results: Left and right AV, respectively, were visualized in 29 (100%) and 22 subjects (76%) by DSA and 29 (100%) and 28 subjects (97%) by SRCTAV, with right-AV detection significantly higher by SRCTAV (p<0.05). Cannulations were regarded successful in 28 subjects having both AVs observed on SRCTAV but not in the remaining subject whose adrenocortical scintigram was positive, however. Adrenalectomy was performed with a diagnosis of adenoma. Among 28 subjects with successful AVS, histopathological diagnoses included adenoma (25), nodular hyperplasia (2) and normal (1). After adrenalectomy, antihypertensive drug usage in 28 patients was reduced or stopped with decreases in SAC (97%)., Conclusions: Detection of AV was significantly higher by SRCTAV than DSA, especially on the right side, in 29 subjects diagnosed with one-sided APT., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objective: Correct interpretation of renal function in patients with primary aldosteronism is difficult before adrenalectomy, because subtle kidney impairment is often masked by glomerular hyperfiltration peculiar to primary aldosteronism. The aim of this study was to investigate postoperative changes in renal function for patients with primary aldosteronism and to identify clinical predictors of chronic kidney disease manifested postoperatively in the patients without pre-existing chronic kidney disease., Methods: Records of 78 Japanese patients who underwent unilateral adrenalectomy for primary aldosteronism were retrospectively surveyed. Patients who had been followed up for <6 months were excluded. Preoperative and postoperative estimated glomerular filtration rate were compared. Furthermore, uni- and multivariate analyses were carried out to identify clinical predictors for chronic kidney disease manifested postoperatively., Results: Patients with preoperative estimated glomerular filtration rate ≥60 mL/min/1.73 m(2) showed a significant decrease after surgery. Of the 66 patients without pre-existing chronic kidney disease, 24 developed chronic kidney disease postoperatively. Multivariate logistic regression analysis identified a medical history of dyslipidemia as an independent predictor for chronic kidney disease manifested postoperatively. According to univariate analyses, additional factors associated with postoperative manifestation of chronic kidney disease included older age, lower diastolic blood pressure and lower estimated glomerular filtration rate., Conclusions: The interpretation of normal or abnormal renal functions by examining estimated glomerular filtration rate heightened by hyperfiltration alone can mislead clinicians before adrenalectomy. Clinicians should pay attention to patients at greater risk of a significant decline in postoperative renal function., (© 2012 The Japanese Urological Association.)

Background: Primary aldosteronism caused by aldosterone-producing adenoma is the most common curable cause of secondary hypertension, but despite resection, many patients continue to require antihypertensive medications to control their blood pressure postoperatively. The Aldosteronoma Resolution Score is a preoperative 4-item predictive model for the complete postoperative resolution of hypertension. Our aim was to validate the accuracy of this model in predicting postoperative resolution of hypertension in Japanese patients., Methods: The records of 91 Japanese patients who underwent unilateral adrenalectomy for aldosterone-producing adenoma were surveyed retrospectively. Patients were distributed into 2 groups according to whether blood pressure was normal without antihypertensive medications at 6 months postoperatively. Clinical and biochemical data were evaluated at baseline and after the 6-month follow-up., Results: At 6 months, blood pressure had normalized in 45% of the patients without antihypertensive medications. Multivariate logistic regression analysis revealed that patients who had complete resolution of hypertension were significantly more likely to have been taking ≤2 antihypertensive medications preoperatively, have a duration of hypertension of <6 years, and be female. The predictive accuracy of the Aldosteronoma Resolution Score was assessed using the area under the curve of receiver operator characteristics analysis. The value of the area under the curve was 0.81., Conclusion: Our external validation revealed that the Aldosteronoma Resolution Score developed using Western data can identify accurately Japanese individuals with aldosterone-producing adenoma who are likely to have complete resolution of hypertension after adrenalectomy., (Copyright © 2012 Mosby, Inc. All rights reserved.)

We identified a p53 target gene, phosphate-activated mitochondrial glutaminase (GLS2), a key enzyme in conversion of glutamine to glutamate, and thereby a regulator of glutathione (GSH) synthesis and energy production. GLS2 expression is induced in response to DNA damage or oxidative stress in a p53-dependent manner, and p53 associates with the GLS2 promoter. Elevated GLS2 facilitates glutamine metabolism and lowers intracellular reactive oxygen species (ROS) levels, resulting in an overall decrease in DNA oxidation as determined by measurement of 8-OH-dG content in both normal and stressed cells. Further, siRNA down-regulation of either GLS2 or p53 compromises the GSH-dependent antioxidant system and increases intracellular ROS levels. High ROS levels following GLS2 knockdown also coincide with stimulation of p53-induced cell death. We propose that GLS2 control of intracellular ROS levels and the apoptotic response facilitates the ability of p53 to protect cells from accumulation of genomic damage and allows cells to survive after mild and repairable genotoxic stress. Indeed, overexpression of GLS2 reduces the growth of tumor cells and colony formation. Further, compared with normal tissue, GLS2 expression is reduced in liver tumors. Thus, our results provide evidence for a unique metabolic role for p53, linking glutamine metabolism, energy, and ROS homeostasis, which may contribute to p53 tumor suppressor function.