Epigenetic Downregulation of FASN in Visceral Adipose Tissue of Insulin Resistant Subjects.

OBJECTIVE: The risk to develop type 2 diabetes increases with the amount of visceral adiposity presumably due to increased lipolysis and subsequent lipid accumulation in visceral organs. However, data describing the molecular regulation of these pathways in humans are rare. We tested if genes of the lipogenic and lipolytic pathways are associated with glucose intolerance independently of obesity in visceral adipose tissue (VAT) of obese subjects. Moreover, we studied DNA methylation of FASN (fatty acid synthase), that catalyses the synthesis of long-chain fatty acids, in VAT of the same subjects and whether it is associated with metabolic traits. SUBJECTS AND METHODS: Visceral adipose tissue biopsies and blood samples were taken from 93 severely obese subjects undergoing bariatric surgery. Subjects were grouped in low HbA1c (L-HbA1c, HbA1c<6.5 %) and high HbA1c (H-HbA1c, HbA1c≥6.5 %) groups and expression of genes from the lipogenic and lipolytic pathways was analysed by TaqMan qPCR. DNA methylation of FASN was quantified by bisulfite-pyrosequencing. RESULTS: FASN expression was downregulated in visceral fat from subjects with high HbA1c (p = 0.00009). Expression of other lipogenetic (SCD, ELOVL6) or lipolytic genes (ADRB3, PNPLA2) and FABP4 was not changed. DNA methylation of FASN was increased at a regulatory ChoRE recognition site in the H-HbA1c-subgroup and correlated negatively with FASN mRNA (r = - 0.302, p = 0.0034) and positively with HbA1c (r = 0.296, p = 0.0040) and blood glucose (r = 0.363, p = 0.0005). CONCLUSIONS: Epigenetic downregulation of FASN in visceral adipose tissue of obese subjects might contribute to limited de novo lipogenesis of important insulin sensitizing fatty acids and could thereby contribute to glucose intolerance and the development of type 2 diabetes independently of obesity.

Maternal Brown Fat Thermogenesis Programs Glucose Tolerance in the Male Offspring.

Environmental temperature is a driving factor in evolution, and it is commonly assumed that metabolic adaptations to cold climates are the result of transgenerational selection. Here, we show in mice that even minor changes in maternal thermogenesis alter the metabolic phenotype already in the next generation. Male offspring of mothers genetically lacking brown adipose tissue (BAT) thermogenesis display increased lean mass and improved glucose tolerance as adults, while females are unaffected. The phenotype is replicated in offspring of mothers kept at thermoneutrality; conversely, mothers with higher gestational BAT thermogenesis produce male offspring with reduced lean mass and impaired glucose tolerance. Running-wheel exercise reverses the offspring's metabolic impairments, pointing to the muscle as target of these fetal programming effects. Our data demonstrate that gestational BAT activation negatively affects metabolic health of the male offspring; however, these unfavorable fetal programming effects may be negated by active lifestyle.

Multi-layered epigenetic regulation of IRS2 expression in the liver of obese individuals with type 2 diabetes.

AIMS/HYPOTHESIS: IRS2 is an important molecular switch that mediates insulin signalling in the liver. IRS2 dysregulation is responsible for the phenomenon of selective insulin resistance that is observed in type 2 diabetes. We hypothesise that epigenetic mechanisms are involved in the regulation of IRS2 in the liver of obese and type 2 diabetic individuals. METHODS: DNA methylation of seven CpG sites was studied by bisulphite pyrosequencing and mRNA and microRNA (miRNA) expression was assessed by quantitative real-time PCR in liver biopsies of 50 obese non-diabetic and 31 obese type 2 diabetic participants, in a cross-sectional setting. Methylation-sensitive luciferase assays and electrophoretic mobility shift assays were performed. Furthermore, HepG2 cells were treated with insulin and high glucose concentrations to induce miRNA expression and IRS2 downregulation. RESULTS: We found a significant downregulation of IRS2 expression in the liver of obese individuals with type 2 diabetes (0.84 ± 0.08-fold change; p = 0.0833; adjusted p value [pa] = 0.0417; n = 31) in comparison with non-diabetic obese participants (n = 50). This downregulation correlated with hepatic IRS2 DNA methylation at CpG5. Additionally, CpG6, which is located in intron 1 of IRS2, was hypomethylated in type 2 diabetes; this site spans the sterol regulatory element binding transcription factor 1 (SREBF1) recognition motif, which likely acts as transcriptional repressor. The adjacent polymorphism rs4547213 (G>A) was significantly associated with DNA methylation at a specificity-protein-1 (SP1) binding site (CpG3). Moreover, DNA methylation of cg25924746, a CpG site located in the shore region of the IRS2 promoter-associated CpG island, was increased in the liver of individuals with type 2 diabetes, as compared with those without diabetes. A second epigenetic mechanism, upregulation of hepatic miRNA hsa-let-7e-5p (let-7e-5p) in obese individuals with type 2 diabetes (n = 29) vs non-diabetic obese individuals (n = 49) (1.2 ± 0.08-fold change; p = 0.0332; pa = 0.0450), is likely to act synergistically with altered IRS2 DNA methylation to decrease IRS2 expression. Mechanistic in vitro experiments demonstrated an acute upregulation of let-7e-5p expression and simultaneous IRS2 downregulation in a liver (HepG2) cell line upon hyperinsulinaemic and hyperglycaemic conditions. CONCLUSIONS/INTERPRETATION: Our study highlights a new multi-layered epigenetic network that could be involved in subtle dysregulation of IRS2 in the liver of individuals with type 2 diabetes. This might lead to fine-tuning of IRS2 expression and is likely to be supplementary to the already known factors regulating IRS2 expression. Thereby, our findings could support the discovery of new diagnostic and therapeutic strategies for type 2 diabetes. Graphical abstract.

Biperiden and mepazine effectively inhibit MALT1 activity and tumor growth in pancreatic cancer.

MALT1 is a key mediator of NF-κB signaling and a main driver of B-cell lymphomas. Remarkably, MALT1 is expressed in the majority of pancreatic ductal adenocarcinomas (PDACs) as well, but absent from normal exocrine pancreatic tissue. Following, MALT1 shows off to be a specific target in cancer cells of PDAC without affecting regular pancreatic cells. Therefore, we studied the impact of pharmacological MALT1 inhibition in pancreatic cancer and showed promising effects on tumor progression. Mepazine (Mep), a phenothiazine derivative, is a known potent MALT1 inhibitor. Newly, we described that biperiden (Bip) is a potent MALT1 inhibitor with even less pharmacological side effects. Thus, Bip is a promising drug leading to reduced proliferation and increased apoptosis in PDAC cells in vitro and in vivo. By compromising MALT1 activity, nuclear translocation of c-Rel is prevented. c-Rel is critical for NF-κB-dependent inhibition of apoptosis. Hence, off-label use of Bip or Mep represents a promising new therapeutic approach to PDAC treatment. Regularly, the Anticholinergicum Bip is used to treat neurological side effects of Phenothiazines, like extrapyramidal symptoms.

Critical evaluation of the DNA-methylation markers ABCG1 and SREBF1 for Type 2 diabetes stratification.

Aim: Validation of epigenome-wide association studies is sparse. Therefore, we evaluated the methylation markers cg06500161 (ABCG1) and cg11024682 (SREBF1) as classifiers for diabetes stratification. Patients & methods: DNA methylation was measured in blood (n = 167), liver (n = 99) and visceral adipose tissue (n = 99) of nondiabetic or Type 2 diabetic subjects by bisulfite pyrosequencing. Results: DNA methylation at cg11024682 in blood and liver correlated with BMI. Methylation at cg06500161 was influenced by the adjacent SNP rs9982016. Insulin-resistant and sensitive subjects could be stratified by DNA methylation status in blood or visceral adipose tissue. Conclusion: DNA methylation at both loci in blood presents a promising approach for risk group stratification and could be valuable for personalized Type 2 diabetes risk prediction in the future.

Reduced expression of thyroid hormone receptor ß in human nonalcoholic steatohepatitis.

Hepatic thyroid hormone signaling has an important role in the development and progression of nonalcoholic steatohepatitis (NASH). While the systemic levels of thyroid hormone might remain stable, there is evidence that the intracellular signaling machinery consisting of transporters, deiodinases and receptors could be altered in NASH. However, clinical material from human liver biopsies of individuals with NASH has not been studied to date. In a cross-sectional study, we analyzed 85 liver biopsies from patients with different stages of NASH that underwent bariatric surgery. Using qPCR, we analyzed gene expression of thyroid hormone transporters NTCP (SLC10A1), MCT8 (SLC16A2) and OATP1C1 (SLCO1C1), thyroid hormone receptor α and ß (THRA and THRB) and deiodinase type I, II and III (DIO1, DIO2, DIO3). The expression was correlated with serum TSH, triglyceride, HbA1c and NASH score and corrected for age or gender if required. While DIO2, DIO3 and SLCO1C1 were not expressed in human liver, we observed a significant negative correlation of THRB and DIO1 with age, and SLC16A2 with gender. THRB expression was also negatively associated with serum triglyceride levels and HbA1c. More importantly, its expression was inversely correlated with NASH score and further declined with age. Our data provide unique insight into the mRNA expression of thyroid hormone transporters, deiodinases and receptors in the human liver. The findings allow important conclusions on the intrahepatic mechanisms governing thyroid hormone action, indicating a possible tissue resistance to the circulating hormone in NASH, which becomes more prominent in advanced age.

Endogenous testosterone and exogenous oxytocin influence the response to baby schema in the female brain.

Nurturing behavior may be critically influenced by the interplay of different hormones. The neuropeptide oxytocin is known to promote maternal behavior and its reduction has been associated with postpartum depression risk and child neglect. Contrariwise, the observed decrease in testosterone level during early parenthood may benefit caretaking behavior, whereas increased testosterone may reduce attention to infants. Here we used functional magnetic resonance imaging to investigate the interactive influence of testosterone and oxytocin on selective attention to and neural processing of the baby schema (BS). 57 nulliparous women performed a target detection task with human faces with varying degree of BS following double-blinded placebo-controlled oxytocin administration in a between-subjects design. Our results support the idea that oxytocin enhances attention to the BS. Oxytocin had a positive effect on activation of the inferior frontal junction during identification of infant targets with a high degree of BS that were presented among adult distractors. Further, activation of the putamen was positively correlated with selective attention to the BS, but only in women with high endogenous testosterone who received oxytocin. These findings provide initial evidence for the neural mechanism by which oxytocin may counteract the negative effects of testosterone in the modulation of nurturing behavior.

Incidental Intraductal Papillary Mucinous Neoplasm, Cystic or Premalignant Lesions of the Pancreas: The Case for Aggressive Management.

Incidental cystic intrapancreatic lesions are daily findings in abdominal radiology. The discovery of incidental pancreatic lesions is increasingly common with technologic diagnostic advancements. This article provides a perspective and guideline on the clinical management of incidental intraductal papillary mucinous neoplasms and cystic or premalignant lesions of the pancreas.

Evaluation of the germline single nucleotide polymorphism rs583522 in the TNFAIP3 gene as a prognostic marker in esophageal cancer.

Most esophageal cancer patients die because of disease relapse, hence an accurate prognosis of disease relapse and survival is essential. Genetic variations in cancer patients may serve as important indicators. Three genotypes (GG, AG, and AA) are displayed by the single nucleotide polymorphism (SNP) rs583522, which maps to the TNFAIP3 gene on chromosome 6. Evaluation of the potential prognostic value of the TNFAIP3-SNP in esophageal cancer (EC) was the aim of this study. A total of 158 patients underwent complete surgical resection of the esophagus for EC. None of them received any neoadjuvant or adjuvant treatment. Peripheral blood was sampled, and genomic DNA was extracted from leukocytes before each operation. Clinicopathologic parameters, tumor cell dissemination in bone marrow, and clinical outcome were correlated with the TNFAIP3-SNP. A-allele carriers showed advanced tumor stages compared with those of homozygous G-allele carriers (P<0.001). Patients with an A-allele genotype (AA or AG) were significantly more likely to experience a relapse (P=0.003). Survival analysis (log-rank test) revealed a significant difference in overall survival between the three groups (P=0.039); however, none of the genotypes was identified as a disease stage-independent prognostic marker. In conclusion, TNFAIP3-SNP stratifies patients into different risk groups; however, it could not be identified as an independent prognostic marker.

COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer.

BACKGROUND: Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties. METHODS: Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data. RESULTS: Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037). CONCLUSION: This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.

Salvage Completion Pancreatectomies as Damage Control for Post-pancreatic Surgery Complications: A Single-Center Retrospective Analysis.

BACKGROUND: Post-pancreatic surgical morbidity is frequent but often manageable by less invasive means than re-operation. Yet, some complications can become hazardous and life threatening. Herein, the results of a completion pancreatectomy (CP) to cope with severe post-operative pancreatic fistulas (POPF) and bleeding complications after major pancreatic resections for suspected pancreatic malignancy are presented. METHODS: CPs to treat severe post-pancreatic index-surgery complications between January 2002 and January 2012 were selected out of a prospective database. Indications for CP as well as perioperative data were prospectively collected and retrospectively assessed. RESULTS: In 20 of 521 Kausch-Whipple Resections (3.8%), a CP was necessary to treat post-index surgery morbidity. Indications included insufficiency of the pancreaticojejunal anastomosis with resulting POPF in 14 (70.0%) patients, severe bleeding complications in 6 (30.0%) patients, and a severe portal vein thrombosis in 1 (5.0%) patient. In 7 (35.0%) of the 20 patients, the course was complicated by remnant pancreatitis. Eleven (55.0%) of the 20 patients died during the hospital stay. Median time to re-operation did not significantly differ between survivors and in-hospital deaths (10.0 vs. 8.0 days; p = 0.732). Median hospital stay of the surviving patients was 31.0 (range 10-113) days. Re-operations following CPs were necessary in 5 (55.6%) of the 9 patients who survived and in 9 (81.8%) out of 11 patients who died. CONCLUSIONS: Post-pancreatic resection complications can become hazardous and result in severely ill patients requiring maximum therapy. CP in these cases has a high mortality but serves as an ultima ratio to cope with deleterious complications.

Cyclin D1 is a strong prognostic factor for survival in pancreatic cancer: analysis of CD G870A polymorphism, FISH and immunohistochemistry.

BACKGROUND AND OBJECTIVE: Cyclin D1 is an important regulator protein for the G1-S cell cycle phase transition. The aim of this trial was to evaluate the impact of the CCND1 polymorphism G870A and corresponding protein expression and CCND1 amplification on the survival of the patients. METHODS: 425 patients with ductal pancreatic adenocarcinoma who underwent resection were included after histopathological confirmation. DNA was analyzed for Cyclin D1 polymorphisms, immunhistochemical examination and fluorescence in situ hybridization analysis of the tumor were performed. RESULTS: Overall, the mean survival was 22.9 months (20.5-25.3). The survival in patients with Cyclin D1 G870A polymorphism Adenine/Adenine was 15.1 months (95% CI 11.3-18.9), 21.5 months (17.4-25.6) for Adenine/Guanine, and 29.4 months (95% CI 23.8-35.0) for Guanine/Guanine (P = 0.003). A shorter survival was associated with strong/moderate protein expression in immunohistochemistry (IHC) compared to weak/no expression (P = 0.028). Additionally, a significant coherency between unfavourable polymorphism (AA/AG) and increased protein expression was detected (P = 0.005). CONCLUSIONS: A strong impact on survival of Cyclin D1 G870A polymorphism and the detected corresponding protein expression was found. The biological mechanism of CCND1 in carcinogenesis has not been fully examined; but at present Cyclin D1 seems to be an interesting biomarker for the prognosis of ductal adenocarcinoma.

An A/C germline single-nucleotide polymorphism in the TNFAIP3 gene is associated with advanced disease stage and survival in only surgically treated esophageal cancer.

Prognostication of disease relapse and survival is essential for cancer patients and genetic variations in cancer patients may serve as important indicators. A single-nucleotide polymorphism (SNP) mapping to the tumor necrosis factor, alpha-induced protein 3 (TNFAIP3) gene at position 138241110 displays three genotypes (AA, AC and CC). The aim of this study was to evaluate the potential prognostic value of the TNFAIP3-SNP in esophageal cancer (EC). Genomic DNA was extracted from peripheral blood leukocytes of 173 patients who underwent complete surgical resection for EC and did not receive any neoadjuvant or adjuvant therapy. For SNP detection, a 260- bp fragment was PCR amplified, purified and sequenced with tested primers. The product was analyzed by automatic DNA sequencer.The TNFAIP3 genotypes were correlated with clinico-pathological parameters, tumor cell dissemination in bone marrow and clinical outcome. The C-allele carrier presented with higher disease stage (P<0.001). This was predominantly because of the presence of lymph node metastasis (P<0.001). The recurrence rate was higher in C-allele carriers (AC and CC genotype; P=0.004). Kaplan-Meier plots for disease-free (P=0.017) and overall survival (P<0.001) displayed a gene dosage-associated outcome with AA genotype patients presenting the longest and CC genotype patients the poorest survival. In disease stage-adjusted multivariate analysis the TNFAIP3-SNP was identified as an independent prognostic factor for survival (hazard ratio 1.9; P=0.008). The TNFAIP3-SNP allows risk stratification of EC patients and may be a useful tool to identify patients eligible for multimodal therapy concepts.

Chromosome 8p deletions and 8q gains are associated with tumor progression and poor prognosis in prostate cancer.

PURPOSE: Deletions of 8p and gains of 8q belong to the most frequent cytogenetic alterations in prostate cancer. The target genes of these alterations and their biological significance are unknown. EXPERIMENTAL DESIGN: To determine the relationship between chromosome 8 changes, and prostate cancer phenotype and prognosis, a set of 1.954 fully annotated prostate cancers were analyzed in a tissue microarray format by fluorescence in situ hybridization. RESULTS: Both 8p deletions and 8q gains increased in number during different stages of prostate cancer progression. 8p deletions/8q gains were found in 26.1%/4.8% of 1,239 pT(2) cancers, 38.5%/9.8% of 379 pT(3a) cancers, 43.5%/8.9% of 237 pT(3b) cancers, 40.7%/14.8% of 27 pT(4) cancers, 39.1%/34.8% of 23 nodal metastases, 51.9%/33.3% of 27 bone metastases, and 45.5%/59.9% of 22 hormone refractory cancers (P < 0.0001 each). Both 8p deletions and 8q gains were also significantly associated with high Gleason grade and with each other (P < 0.0001 each). In primary tumors, 8p deletions were seen in only 27.3% of 1,882 cancers without 8q gain but in 57.4% of 122 tumors with 8q gain (P < 0.0001). Among cancers treated with radical prostatectomy, 8p deletions (P = 0.003) and 8q gains (P = 0.02) were associated with biochemical tumor recurrence. However, multivariate analysis (including prostate-specific antigen, pT/pN stage, Gleason score, and surgical margin status) did not reveal any statistically independent effect of 8p or 8q alterations on biochemical tumor recurrence. CONCLUSIONS: 8p deletions and 8q gains are relatively rare in early stage prostate cancer but often develop during tumor progression. The prognostic effect does not seem to be strong enough to warrant clinical application.