Clarissa Silva Martins

A Novel ADIPOQ Mutation (p.M40K) Impairs Assembly of High-Molecular-Weight Adiponectin and Is Associated With Early-Onset Obesity and Metabolic Syndrome

CONTEXT The phenotypic effects of ADIPOQ mutations early in life, prior to type 2 diabetes onset, have not been studied. AIM The objective of the study was to characterize the impact of a novel ADIPOQ mutation in vitro and in vivo. DESIGN The design of the study was ADIPOQ screening, adiponectin oligomerization, and cardiometabolic phenotype assessment. SUBJECTS Fourteen hypoadiponectinemic (<3 μg/mL) and 686 normoadiponectinemic young adults (23-25 y) were prospectively followed up since birth. MAIN OUTCOME MEASURES Human and recombinant murine mutant adiponectin oligomerization, the probands ADIPOQ and ADIPOR1/R2 adipose tissue (AT) expression, and cardiometabolic profile were measured. RESULTS The heterozygous ADIPOQ p.M40K mutation was identified in one hypoadiponectinemic male (2.4 μg/mL) and three other family members. Carriers presented a marked reduction of serum high-molecular weight to total adiponectin ratio when compared with controls (9.4% ± 1% vs 56.6% ± 13%; P < .05) and family noncarriers (9.4% ± 1% vs 42% ± 0.5%; P = .05). Both mRNA and protein levels of adiponectin were increased in the AT of the proband (2.3- and 1.6-fold, respectively). However, the high-molecular weight to total adiponectin ratio of adiponectin was decreased (3.3-fold). Moreover, the expressions of ADIPOR1 and ADIPOR2 were significantly down-regulated in the AT of the proband (6- and 1.2-fold, respectively). The results were confirmed by in vitro studies on the recombinant murine homologous mutation (p.M43K). The probands cardiometabolic phenotype progression was further characterized: born small for gestational age and adolescence-onset obesity; insulin resistance (homeostasis assessment model of insulin resistance of 4.7), and dyslipidemia at 25 years; decreased high-molecular weight adiponectin (0.24 μg/mL = 10%), hypertension (180/120 mm Hg), steatosis (fat liver = 40% ± 6%), increased carotid intima-media thickness at 31 years, and type 2 diabetes (glycosylated hemoglobin = 6.6%) at 34 years of age. Of note, all of the affected family members presented features of metabolic syndrome. CONCLUSION The newly identified ADIPOQ p.M40K mutation associates with severe cardiometabolic dysfunction due to the impairment of high-molecular weight adiponectin complex formation.

NR3C1 polymorphisms in Brazilians of Caucasian, African, and Asian ancestry: glucocorticoid sensitivity and genotype association

OBJECTIVE The Brazilian population has heterogeneous ethnicity. No previous study evaluated NR3C1 polymorphisms in a Brazilian healthy population. MATERIALS AND METHODS We assessed NR3C1 polymorphisms in Brazilians of Caucasian, African and Asian ancestry (n = 380). In a subgroup (n = 40), we compared the genotypes to glucocorticoid (GC) sensitivity, which was previously evaluated by plasma (PF) and salivary (SF) cortisol after dexamethasone (DEX) suppression tests, GC receptor binding affinity (K d ), and DEX-50% inhibition (IC 50 ) of concanavalin-A-stimulated mononuclear cell proliferation. p.N363S (rs6195), p.ER22/23EK (rs6189-6190), and BclI (rs41423247) allelic discrimination was performed by Real-Time PCR (Polymerase Chain Reaction). Exons 3 to 9 and exon/intron boundaries were amplified by PCR and sequenced. RESULTS Genotypic frequencies (%) were: rs6195 (n = 380; AA:96.6/AG:3.14/GG:0.26), rs6189-6190 (n = 264; GG:99.6/GA:0.4), rs41423247 (n = 264; CC:57.9/CG:34.1/GG:8.0), rs6188 (n = 155; GG:69.6/GT:25.7/TT:4.7), rs258751 (n = 150; CC:88.0/CT:10.7/TT:1.3), rs6196 (n = 176; TT:77.2/TC:20.4/CC:2.4), rs67300719 (n = 137; CC:99.3/CT:0.7), and rs72542757 (n = 137; CC:99.3/CG:0.7). The rs67300719 and rs72542757 were found only in Asian descendants, in whom p.N363S and p.ER22/23EK were absent. The p.ER22/23EK was observed exclusively in Caucasian descendants. Hardy-Weinberg equilibrium was observed, except in the Asian for rs6188 and rs258751, and in the African for p.N363S. The K d , IC 50 , baseline and after DEX PF or SF did not differ between genotype groups. However, the mean DEX dose that suppressed PF or SF differed among the BclI genotypes (P = 0.03). DEX dose was higher in GG- (0.7 ± 0.2 mg) compared to GC- (0.47 ± 0.2 mg) and CC-carriers (0.47 ± 0.1 mg). CONCLUSION The genotypic frequencies of NR3C1 polymorphisms in Brazilians are similar to worldwide populations. Additionally, the BclI polymorphism was associated with altered pituitary-adrenal axis GC sensitivity.

Transcriptome Analysis Showed a Differential Signature between Invasive and Non-invasive Corticotrophinomas

ACTH-dependent hypercortisolism caused by a pituitary adenoma [Cushing’s disease (CD)] is the most common cause of endogenous Cushing’s syndrome. CD is often associated with several morbidities, including hypertension, diabetes, osteoporosis/bone fractures, secondary infections, and increased cardiovascular mortality. While the majority (≈80%) of the corticotrophinomas visible on pituitary magnetic resonance imaging are microadenomas (MICs, <10 mm of diameter), some tumors are macroadenomas (MACs, ≥10 mm) with increased growth potential and invasiveness, exceptionally exhibiting malignant demeanor. In addition, larger and invasive MACs are associated with a significant increased risk of local complications, such as hypopituitarism and visual defects. Given the clinical and molecular heterogeneity of corticotrophinomas, the aim of this study was to investigate the pattern of genetic differential expression between MIC and MAC, including the invasiveness grade as a criterion for categorizing these tumors. In this study, were included tumor samples from patients with clinical, laboratorial, radiological, and histopathological diagnosis of hypercortisolism due to an ACTH-producing pituitary adenoma. Differential gene expression was studied using an Affymetrix microarray platform in 12 corticotrophinomas, classified as non-invasive MIC (n = 4) and MAC (n = 5), and invasive MAC (n = 3), according to modified Hardy criteria. Somatic mutations in USP8 were also investigated and mutations were identified in six cases. Differential expression analysis demonstrated that non-invasive MIC and MAC have a similar genetic signature, while invasive MACs exhibited a differential expression profile. Among the genes differentially expressed, we highlighted CCND2, ZNF676, DAPK1, and TIMP2, and their differential expression was validated through quantitative real-time PCR in another cohort of 15 non-invasive and 3 invasive cortocotrophinomas. We also identified potential biological pathways associated with growth and invasiveness, TGF-β and G protein signaling pathways, DNA damage response pathway, and pathways associated with focal adhesion. Our study revealed a differential pattern of genetic signature in a subgroup of MAC, supporting a genetic influence on corticotrophinomas in patients with CD.

HPA axis dysregulation, NR3C1 polymorphisms and glucocorticoid receptor isoforms imbalance in metabolic syndrome

Metabolic syndrome (MetS) shares several similarities with hypercortisolism.

The growth hormone receptor exon 3 polymorphism is not associated with height or metabolic traits in healthy young adults

CONTEXT The GHR polymorphisms contribution to the interindividual variability in prenatal and postnatal growth as well as to metabolic traits is controversial. OBJECTIVE The aim of this study is to analyze the association of the GHRfl/d3 polymorphism with prenatal and postnatal growth and metabolic outcomes in adult life and to compare the genotype distribution in different populations. DESIGN 385 community healthy subjects followed from birth to adult life (23-25years old) were grouped according to birth size: small-SGA (n=130, 62 males), appropriate-AGA (n=162, 75 males) and large for gestational age-LGA (n=93, 48 males). GHRfl/d3 genotype distribution and its potential association with anthropometric (at birth, childhood and adult life) and metabolic features (in adult life) were analyzed and compared with data obtained from a systematic review of GHRfl/d3 association studies (31 articles). RESULTS The frequency of the GHR d3/d3 genotype was lower in the LGA (χ2 p=0.01); SGA and AGA subjects exhibited an increased chance of the d3/d3 genotype (OR=3.58; 95%CI: 1.55; 8.24) and (OR=2.39; 95%CI: 1.02; 5.62), respectively. Despite the different prevalence among different birth size groups, in adults, GHRfl/d3 genotype was not associated with height, plasma IGF1 levels or metabolic phenotype and cardiovascular risk. GHRfl/d3 genotype distributions in AGA, SGA and LGA groups were comparable with those found in subjects of European origin but not with those of Asian ancestry. CONCLUSIONS The GHRd3 genotype was negatively associated with birth size but it was not associated with adult height or weight, plasma IGF1, metabolic phenotype or any marker of increased cardiovascular risk in young adults.

Restricted Feeding Schedules Modulate in a Different Manner the Expression of Clock Genes in Rat Hypothalamic Nuclei.

Food access restriction is associated to changes in gene expression of the circadian clock system. However, there are only a few studies investigating the effects of non-photic synchronizers, such as food entrainment, on the expression of clock genes in the central oscillators. We hypothesized that different feeding restriction patterns could modulate the expression of clock genes in the suprachiasmatic nucleus (SCN) “master” clock and in extra-SCN oscillators such as the paraventricular (PVN) and arcuate (ARC) hypothalamic nuclei. Wistar rats were divided into four groups: Control group (CG; food available ad libitum), Restricted night-fed (RF-n; food access during 2 h at night), Restricted day-fed (RF-d; food access during 2 h at daytime), Day-fed (DF; food access during 12 h at daytime). After 21 days, rats were decapitated between ZT2-ZT3 (0800–0900 h); ZT11-ZT12 (1700–1800 h), or ZT17-18 (2300–2400 h). Plasma corticosterone was measured by radioimmunoassay (RIA). The expression of Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, and Rorα were assessed in SCN, PVN, and ARC hypothalamic nuclei by RT-PCR and calculated by the 2[−DeltaDeltaCT(Cyclethreshold)](2−ΔΔCT) method. Restricted food availability during few h led to decreased body weight in RF-n and RF-d groups compared to controls and DF group. We also observed an anticipatory corticosterone peak before food availability in RF-n and RF-d groups. Furthermore, the pattern of clock gene expression in response to RF-n, RF-d, and DF schedules was affected differently in the SCN, PVN, and ARC hypothalamic nuclei. In conclusion, the master oscillator in SCN as well as the oscillator in PVN and ARC, all brain areas involved in food intake, responds in a tissue-specific manner to feeding restriction.

SAGE analysis highlights the putative role of underexpression of ribosomal proteins in GH-secreting pituitary adenomas.

BACKGROUND Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis. OBJECTIVE The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression. METHODS Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries. RESULTS Computer-generated genomic analysis tools identified 13,722 and 14,993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR. CONCLUSION We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.

Absence of TERT promoter mutations in pituitary adenomas

Ethical approval All procedures performed involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments. This study was approved by the Institutional Review Board of the University Hospital of the Ribeirao Preto Medical School, University of Sao Paulo, Brazil (Process n° 11071/2013).

Impact of the Canonical Wnt Pathway Activation on the Pathogenesis and Prognosis of Adamantinomatous Craniopharyngiomas

CTNNB1 mutations and abnormal β-catenin distribution are associated with the pathogenesis of adamantinomatous craniopharyngioma (aCP). We evaluated the expression of the canonical Wnt pathway components in aCPs and its association with CTNNB1 mutations and tumor progression. Tumor samples from 14 aCP patients and normal anterior pituitary samples from eight individuals without pituitary disease were studied. Gene expression of Wnt pathway activator (WNT4), inhibitors (SFRP1, DKK3, AXIN1, and APC), transcriptional activator (TCF7), target genes (MYC, WISP2, and, CDH1), and Wnt modulator (TP53) was evaluated by qPCR. β-Catenin, MYC, and WISP2 expression was determined by immunohistochemistry (IHC). The transcription levels of all genes studied, except APC, were higher in aCPs as compared to controls and TCF7 mRNA levels correlated with CTNNB1 mutation. CDH1 mRNA was overexpressed in tumor samples of patients with disease progression in comparison to those with stable disease. β-Catenin was positive and aberrantly distributed in 11 out of 14 tumor samples. Stronger β-catenin immunostaining associated positively with tumor progression. MYC positive staining was found in 10 out of 14 cases, whereas all aCPs were negative for WISP2. Wnt pathway genes were overexpressed in aCPs harboring CTNNB1 mutations and in patients with progressive disease. Recurrence was associated with stronger staining for β-catenin. These data suggest that Wnt pathway activation contributes to the pathogenesis and prognosis of aCPs.

Letter to the Editor: Comments on “LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal” by Shaikh L.H., et al

In the analysis of the transcriptome of zona glomerulosa and zona fasciculata of adrenal tissue adjacent to aldosteroneproducing adenomas or pheochromocytomas, Shaikh et al (1) found that leucine-rich repeat-containing G proteincoupled receptor 5 (LGR5), the gene encoding a cell surface E3 ubiquitin ligase related to the Wnt noncanonical pathway, was the most expressed gene in human zona glomerulosa and was almost absent in adjacent aldosteroneproducing adenomas. In functional assays, using cell line or primary cell cultures, the investigators also found that stimulation of the LRG5 (LGR5 transfection or R-spondin-3 stimulation) inhibited aldosterone production and provoked a reduction in the number of cells in culture. LGR5 transfection also increased activator protein-1/Jun activity, suggesting that the activation of a noncanonical Wnt signaling pathway is the mediator of the LGR5 response. Prior to the present work, we reported, for the first time, the overexpression of MAPK8, an effector of the noncanonical Wnt/planar cell polarity (Wnt/PCP), wingless-type MMTV integration site family, member 5 (WNT5A), and nuclear factor of activated T cells, the ligand and the effector, respectively, of the noncanonical Wnt/calcium (Wnt/Ca) pathway, and the underexpression of prickle homolog 1 (PRICKLE), an inhibitor of the Wnt/PCP pathway in adult and pediatric adrenocortical tumors as compared with normal adrenal tissues (2). We also found an association between Wnt/PCP and Wnt/Ca noncanonical pathway activation with poor outcomes of these tumors, which would suggest a putative involvement in tumor aggressiveness. Additionally, our group studying in NCI-H295 cells the effect of PNU74654, which acts as a Wnt canonical antagonist by preventing Tcf from binding to -catenin, reported a markedly decrease in the relative protein expression of aldosterone synthase (3). The innovative data from Shaikh et al (1) combined with the findings from our group support that both canonical and noncanonical Wnt pathways contribute to adrenocortical tumorigenesis and aldosterone production. The authors chose not to comment on this letter.