A Prospective Randomized Controlled Trial of the Metabolic Effects of Sleeve Gastrectomy with Transit Bipartition.

PURPOSE: To compare the effects of the sleeve gastrectomy with transit bipartition (SG + TB) procedure with standard medical therapy (SMT) in mildly obese patients with type II diabetes (T2D). METHODS: This is a prospective, randomized, controlled trial. Twenty male adults, ≤ 65 years old, with T2D, body mass index (BMI) > 28 kg/m2 and < 35 kg/m2, and HbA1c level > 8% were randomized to SG + TB or to SMT. Outcomes were the remission in the metabolic and cardiovascular risk variables up to 24 months. RESULTS: At 24 months, SG + TB group showed a significant decrease in HbaA1c values (9.3 ± 2.1 versus 5.5 ± 1.1%, P = < 0.05) whereas SMT group maintained similar levels from baseline (8.0 ± 1.5 versus 8.3 ± 1.1%, P = NS). BMI values were lower in the SG + TB group (25.3 ± 2.8 kg/m2 versus 30.9 ± 2.5 kg/m2; P = < 0.001). At 24 months, none patient in SG + TB group needed medications for hyperlipidemia/hypertension. HDL-cholesterol levels increased in the SG + TB group (33 ± 8 to 45 ± 15 mg/dL, P < 0.001). After 24 months, the area under the curve (AUC) of GLP1 increased and in the SG + TB group and the AUC of the GIP concentrations was lower in the SG + TB group than in the SMT. At 3 months, SG + TB group showed a marked increase in FGF19 levels (74.1 ± 45.8 to 237.3 ± 234 pg/mL; P = 0.001). CONCLUSIONS: SG + TB is superior to SMT and was associated with a better metabolic and cardiovascular profile.

Tissue-specific methylation profile in obese patients with type 2 diabetes before and after Roux-en-Y gastric bypass.

Eating habits, lifestyles, and exposure to specific environmental factors can greatly impact the risk of developing type 2 diabetes (T2D), influence the genome epigenetically, and affect the expression of genes, including genes related to glycemic control, at any stage of life. The epigenetic mechanism underlying obesity and T2D pathogenesis remains poorly understood. Conventional strategies for the treatment of obesity and its comorbidities often have poor long-term adherence, and pharmacological interventions are limited. Bariatric surgery is the most effective current option to treat severe obesity, and Roux-en-Y gastric bypass (RYGB) is the most applied technique worldwide. Epigenetic changes differ depending on the approach used to treat obesity and its associated comorbidities (clinical or surgical). Compared to primary clinical care, bariatric surgery leads to much greater loss of body weight and higher remission rates of T2D and metabolic syndrome, with methylation profiles in promoter regions of genes in obese individuals becoming similar to those of normal-weight individuals. Bariatric surgery can influence DNA methylation in parallel with changes in gene expression pattern. Changes in clinical biomarkers that reflect improvements in glucose and lipid metabolism after RYGB often occur before major weight loss and are coordinated by surgery-induced changes in intestinal hormones. Therefore, the intestine methylation profile would assist in understanding the mechanisms involved in improved glycemic control after bariatric surgery. The main objectives in this area for the future are to identify epigenetic marks that could be used as early indicators of metabolic risk, and to develop treatments able to delay or even reverse these epigenetic changes. Studies that provide the "human epigenetic profile" will be of considerable value to identify tissue-specific epigenetic signatures and their role in the development of chronic diseases. Further studies should apply methods based on global analysis of the genome to identify methylated sites associated with disease and epigenetic marks associated with the remodeling response to bariatric surgery. This review describes the main epigenetic alterations associated with obesity and T2D and the potential role of RYGB in remodeling these changes.

Type 2 Diabetes Remission After Roux-en-Y Gastric Bypass: Evidence for Increased Expression of Jejunal Genes Encoding Regenerating Pancreatic Islet-Derived Proteins as a Potential Mechanism.

BACKGROUND: Intestinal expression of regenerating pancreatic islet-derived protein-encoding genes (REG) would be enhanced after Roux-en-Y gastric bypass (RYGB) and would affect postoperative type 2 diabetes remission (T2Dr). METHODS: Intestinal biopsy samples were collected from 20 adult obese women with T2D before and 3 months after RYGB. Levels of REG expression and the gene encoding its putative receptor (EXTL3) were assessed by microarray and validated by quantitative RT-PCR. T2Dr was assessed according to ADA criteria 1 year after RYGB. RESULTS: After RYGB, only patients with T2Dr had significantly increased REG1α and REG3γ expression in the jejunum, as validated by quantitative RT-PCR. CONCLUSIONS: Our data provide support for the hypothesis that increased jejunal expression of REG genes after RYGB affects T2Dr, possibly by playing an endocrine function.

The SURMetaGIT study: Design and rationale for a prospective pan-omics examination of the gastrointestinal response to Roux-en-Y gastric bypass surgery.

Objective To describe the protocol of the SURgically induced Metabolic effects on the Human GastroIntestinal Tract (SURMetaGIT) study, a clinical pan-omics study exploring the gastrointestinal tract as a central organ driving remission of type 2 diabetes mellitus (T2DM) after Roux-en-Y gastric bypass (RYGB). The main points considered in the study's design and challenges faced in its application are detailed. Methods This observational, longitudinal, prospective study involved collection of gastrointestinal biopsy specimens, faeces, urine, and blood from 25 obese women with T2DM who were candidates for RYGB (20 patients for omics assessment and 5 for omics validation). These collections were performed preoperatively and 3 and 24 months postoperatively. Gastrointestinal transcriptomics; faecal metagenomics and metabolomics; plasma proteomics, lipidomics, and metabolomics; and biochemical, nutritional, and metabolic data were assessed to identify their short- and long-term correlations with T2DM remission. Results Data were collected from 20 patients before and 3 months after RYGB. These patients have nearly completed the 2-year follow-up assessments. The five additional patients are currently being selected for omics data validation. Conclusion The multi-integrated pan-omics approach of the SURMetaGIT study enables integrated analysis of data that will contribute to the understanding of molecular mechanisms involved in T2DM remission after RYGB.

Differential expression of genes encoding proteins of the HGF/MET system in insulinomas.

BACKGROUND: Insulinomas are the most common functional pancreatic neuroendocrine tumors, whereas histopathological features do not predict their biological behaviour. In an attempt to better understand the molecular processes involved in the tumorigenesis of islet beta cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two inhibitors of HGF activator and of matriptase). METHODS: Quantitative real-time reverse transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes in 24 sporadic insulinomas: 15 grade 1 (G1), six grade 2 (G2) and three hepatic metastases. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19. RESULTS: Overexpression of MET was observed in the three hepatic metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. A positive correlation was observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene. CONCLUSION: The final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoral progression and metastatization in insulinomas.

In type 2 diabetes mellitus glycated albumin alters macrophage gene expression impairing ABCA1-mediated cholesterol efflux.

Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2.

Relationship between gut hormones and glucose homeostasis after bariatric surgery.

Type 2 diabetes mellitus (T2D) is emerging as a worldwide public health problem, and is mainly associated with an increased incidence of obesity. Bariatric surgery is currently considered the most effective treatment for severely obese patients. After bariatric surgery, T2D patients have shown a significant improvement in glycemic control, even before substantial weight loss and often discontinuation of medication for diabetes control. A central role for enteroendocrine cells from the epithelium of the gastrointestinal tract has been speculated in this postoperative phenomenon. These cells produce and secrete polypeptides - gut hormones - that are associated with regulating energy intake and glucose homeostasis through modulation of peripheral target organs, including the endocrine pancreas. This article reviews and discusses the biological actions of the gut hormones ghrelin, cholecystokinin, incretins, enteroglucagon, and Peptide YY, all of which were recently identified as potential candidates for mediators of glycemic control after bariatric surgery. In conclusion, current data reinforce the hypothesis that T2D reversion after bariatric surgery may be related to glycemic homeostasis developed by the intestine.

Dual effect of advanced glycation end products in pancreatic islet apoptosis.

BACKGROUND: Loss of ß-cell function hastens deterioration of metabolic control in type 2 diabetes patients. Besides amyloid deposit and glucolipotoxicity, advanced glycation end products (AGEs) acting through their receptors (RAGE) seem to contribute to this process by promoting islet apoptosis. In order to investigate the role of AGEs in ß-cell deterioration, we evaluated the temporal and dose effects of AGE compounds on apoptosis rate, reactive oxygen species generation and expression of pro-apoptotic and anti-apoptotic genes in cultured islets. METHODS: Rat pancreatic islets were exposed or not for 24, 48, 72 and 96 h to albumin modified by glycoaldehyde. Apoptosis, reactive oxygen species and superoxide content and NADPH oxidase activity were evaluated as well as RNA expression of the genes Ager (codes for RAGE), Bax, Bcl2 and Nfkb1. RESULTS: In 24 and 48 h, glycoaldehyde elicited a decrease in apoptosis rate in comparison with the control condition concomitantly with a reduction in Bax/Bcl2 RNA ratio and in Nfkb1 RNA expression. In contrast, after 72 and 96 h, glycoaldehyde promoted an increase in apoptosis rate concomitantly with an increase in Bax/Bcl2 RNA ratio and in Nfkb1 RNA expression. In 24 h, glycoaldehyde elicited a decrease in the islet content of reactive oxygen species, whereas after 48 and 72 h, it promoted an opposite effect, increasing superoxide generation. The NADPH oxidase inhibitor VAS2870 attenuated superoxide production, implicating NADPH oxidase as an important source of reactive oxygen species in islets exposed to AGEs. CONCLUSIONS: Albumin modified by glycoaldehyde exerted a dual effect in cultured pancreatic islets, being protective against apoptosis after short exposure but pro-apoptotic after prolonged exposure.

Advanced glycated albumin isolated from poorly controlled type 1 diabetes mellitus patients alters macrophage gene expression impairing ABCA-1-mediated reverse cholesterol transport.

BACKGROUND: We evaluated the effects of albumin isolated from control individuals and from patients with poorly controlled type 1 diabetes mellitus on macrophage gene expression and on reverse cholesterol transport. METHODS: Serum albumin was purified from control subjects (n = 12) and from patients with poorly controlled type 1 diabetes mellitus (n = 13). (14)C-cholesterol-labelled J774 macrophages treated with albumin were employed to measure cholesterol efflux mediated by apo A-I, HDL(3) or HDL(2), the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyse gene expression. Several differentially expressed genes were validated by real-time reverse transcription-PCR using TaqMan Two Step RT-PCR. RESULTS: Levels of glycation-modified and (carboxymethyl)lysine-modified albumin were higher in diabetic patients than in control subjects. Apo A-I-mediated and HDL(2)-mediated cellular cholesterol efflux were impaired in macrophages treated with albumin from diabetic patients in comparison with control albumin-treated cells, which was attributed to the reduction in ABCA-1 protein content. Even in the presence of cholesterol acceptors, a higher level of intracellular lipid was observed in macrophages exposed to albumin from diabetic individuals in comparison with the control. The reduction in ABCA-1 content was associated with enhanced expression of stearoyl CoA desaturase 1 and decreased expression of janus kinase 2, which were induced by albumin from patients with type 1 diabetes mellitus. CONCLUSIONS: (Carboxymethyl)lysine-modified albumin isolated from poorly controlled type 1 diabetic patients impairs ABCA-1-mediated reverse cholesterol transport and elicits intracellular lipid accumulation, possibly contributing to atherosclerosis.

Prevalence of celiac disease among blood donors in São Paulo: the most populated city in Brazil.

OBJECTIVE: Celiac disease is a permanent enteropathy caused by the ingestion of gluten, which leads to an immunemediated inflammation of the small intestine mucosa. The prevalence of celiac disease varies among different nations and ethnic backgrounds, and its diversity is determined by genetic and environmental factors. São Paulo city is one of the largest cities in the world, with a vast population and an important history of internal migratory flow from other Brazilian regions, as well as immigration from other, primarily European, countries, resulting in significant miscegenation. The aim of the present study was to estimate the prevalence of adults with undiagnosed celiac disease among blood donors of São Paulo by collecting information on the ancestry of the population studied. METHODS: The prevalence of celiac disease was assessed by screening for positive IgA transglutaminase and IgA endomysium antibodies in 4,000 donors (volunteers) in the Fundação Pró-Sangue Blood Center of São Paulo, São Paulo, Brazil. The antibody-positive subjects were asked to undergo a small bowel biopsy. RESULTS: Of the 4,000 subjects, twenty-four had positive tests, although both antibody tests were not always concordant. For example, ten subjects were positive for IgA tissue transglutaminase only. In twenty-one positive patients, duodenal biopsies were performed, and the diagnosis of celiac disease was confirmed in fourteen patients (Marsh criteria modified by Oberhuber). In this group, 67% claimed to have European ancestry, mainly from Italy, Portugal and Spain. CONCLUSION: The prevalence of celiac disease is at least 1:286 among supposedly healthy blood bank volunteers in São Paulo, Brazil.

Prevalence of celiac disease among blood donors in São Paulo: the most populated city in Brazil

OBJECTIVE: Celiac disease is a permanent enteropathy caused by the ingestion of gluten, which leads to an immunemediated inflammation of the small intestine mucosa. The prevalence of celiac disease varies among different nations and ethnic backgrounds, and its diversity is determined by genetic and environmental factors. São Paulo city is one of the largest cities in the world, with a vast population and an important history of internal migratory flow from other Brazilian regions, as well as immigration from other, primarily European, countries, resulting in significant miscegenation. The aim of the present study was to estimate the prevalence of adults with undiagnosed celiac disease among blood donors of São Paulo by collecting information on the ancestry of the population studied. METHODS: The prevalence of celiac disease was assessed by screening for positive IgA transglutaminase and IgA endomysium antibodies in 4,000 donors (volunteers) in the Fundação Pró-Sangue Blood Center of São Paulo, São Paulo, Brazil. The antibody-positive subjects were asked to undergo a small bowel biopsy. RESULTS: Of the 4,000 subjects, twenty-four had positive tests, although both antibody tests were not always concordant. For example, ten subjects were positive for IgA tissue transglutaminase only. In twenty-one positive patients, duodenal biopsies were performed, and the diagnosis of celiac disease was confirmed in fourteen patients (Marsh criteria modified by Oberhuber). In this group, 67% claimed to have European ancestry, mainly from Italy, Portugal and Spain. CONCLUSION: The prevalence of celiac disease is at least 1:286 among supposedly healthy blood bank volunteers in São Paulo, Brazil.

Association of genetic variants in the promoter region of genes encoding p22phox (CYBA) and glutamate cysteine ligase catalytic subunit (GCLC) and renal disease in patients with type 1 diabetes mellitus.

BACKGROUND: Oxidative stress is recognized as a major pathogenic factor of cellular damage caused by hyperglycemia. NOX/NADPH oxidases generate reactive oxygen species and NOX1, NOX2 and NOX4 isoforms are expressed in kidney and require association with subunit p22phox (encoded by the CYBA gene). Increased expression of p22phox was described in animal models of diabetic nephropathy. In the opposite direction, glutathione is one of the main endogenous antioxidants whose plasmatic concentrations were reported to be reduced in diabetes patients. The aim of the present investigation was to test whether functional single nucleotide polymorphisms (SNPs) in genes involved in the generation of NADPH-dependent O2•⁻ (-675 T → A in CYBA, unregistered) and in glutathione metabolism (-129 C → T in GCLC [rs17883901] and -65 T → C in GPX3 [rs8177412]) confer susceptibility to renal disease in type 1 diabetes patients. METHODS: 401 patients were sorted into two groups according to the presence (n = 104) or absence (n = 196) of overt diabetic nephropathy or according to glomerular filtration rate (GFR) estimated by Modification of Diet in Renal Disease (MDRD) equation: ≥ 60 mL (n = 265) or < 60 mL/min/1.73 m² (n = 136) and were genotyped. RESULTS: No differences were found in the frequency of genotypes between diabetic and non-diabetic subjects. The frequency of GFR < 60 mL/min was significantly lower in the group of patients carrying CYBA genotypes T/A+A/A (18.7%) than in the group carrying the T/T genotype (35.3%) (P = 0.0143) and the frequency of GFR < 60 mL/min was significantly higher in the group of patients carrying GCLC genotypes C/T+T/T (47.1%) than in the group carrying the C/C genotype (31.1%) (p = 0.0082). Logistic regression analysis identified the presence of at least one A allele of the CYBA SNP as an independent protection factor against decreased GFR (OR = 0.38, CI95% 0.14-0.88, p = 0.0354) and the presence of at least one T allele of the GCLC rs17883901 SNP as an independent risk factor for decreased GFR (OR = 2.40, CI95% 1.27-4.56, p = 0.0068). CONCLUSIONS: The functional SNPs CYBA -675 T → A and GCLC rs17883901, probably associated with cellular redox imbalances, modulate the risk for renal disease in the studied population of type 1 diabetes patients and require validation in additional cohorts.

Acromegaly: correlation between expression of somatostatin receptor subtypes and response to octreotide-lar treatment.

About one-third of acromegalics are resistant to the clinically available somatostatin analogs (SA). The resistance is related to density reduction or different expression of somatostatin receptor subtypes (SSTR). This study analyzes SSTR's expression in somatotrophinomas, comparing to SA response, hormonal levels, and tumor volume. We analyzed 39 somatotrophinomas; 49% were treated with SA. The most expressed SSTR was SSTR5, SSTR3, SSTR2, SSTR1, and SSTR4, respectively. SSTR1 and SSTR2 had higher expression in patients that had normalized GH and IGF-I. SSTR3 was more expressed in patients with tumor reduction. There was a positive correlation between the percentage of tumor reduction and SSTR1, SSTR2 and SSTR3 expression. Also, a positive correlation between SSTR2 mRNA expression and the immunohistochemical reactivity of SSTR2 was found. Our study confirmed the association between the SA response to GH and IGF-I and the SSTR2. Additionally, this finding was also demonstrated in relation to SSTR1.

Genes differentially expressed in prolactinomas responsive and resistant to dopamine agonists.

BACKGROUND/AIMS: Prolactin (PRL) secretion and its gene expression are inhibited by dopamine. Prolactinomas are the most common secreting pituitary adenomas, and dopamine agonists (DA) are the first choice for their treatment. However, a subset of patients is resistant to DA. As the mechanisms involved in DA resistance are not fully understood, the aim of this study was to obtain new insights regarding the molecular differences between the prolactinomas that are responsive to DA and those that are resistant. METHODS: Tumor tissue samples were collected from 17 patients who harbored prolactinomas, which were classified as responsive or resistant according to their clinical and laboratorial reaction to DA. The expression of 6 genes was evaluated by real-time polymerase chain reaction: dopamine receptor type 2 (DRD(2)), nerve growth factor-beta (NGFB) and its receptor (NGFR), estrogen receptor-alpha (ERA), estrogen receptor-beta (ERB) and the pituitary tumor transforming gene (PTTG). RESULTS: Median DRD(2) and NGFR expression in responsive patients was significantly higher than in resistant ones (p = 0.029 and p = 0.020, respectively). Moreover, the expressions of DRD(2) and NGFR were positively correlated with PRL decrease during treatment (r = 0.66, p = 0.005 and r = 0.57, p = 0.044, respectively). Furthermore, ERB expression was positively correlated to PTTG expression (r = 0.68, p = 0.032) and negatively correlated to NGFB expression (r = -0.75, p = 0.02). CONCLUSIONS: DRD(2) and NGFR expressions are related to the responsiveness of prolactinoma to DA. However, PTTG, ERB and ERA expressions are not. Also ERB, ERA and PTTG expressions did not present a clear correlation to tumor aggressiveness. Furthermore, the response of prolactinomas to DA should be viewed as a spectrum ranging from the most responsive to the most resistant ones.

Association between tumoral GH-releasing peptide receptor type 1a mRNA expression and in vivo response to GH-releasing peptide-6 in ACTH-dependent Cushing's syndrome patients.

OBJECTIVE: GH secretagogues (GHS) produce exaggerated ACTH and cortisol responses in Cushing's disease (CD) patients, attributable to their direct action on GH-releasing peptide receptor type 1a (GHSR-1a). However, there are no studies correlating the in vivo response to GHS and GHSR-1a mRNA expression in ACTH-dependent Cushing's syndrome (CS) patients. The aim of this study is to correlate the patterns of ACTH and cortisol response to GH-releasing peptide-6 (GHRP-6) to GHSR-1a expression in ACTH-dependent CS patients. DESIGN: Prospective study in a tertiary referral hospital center. Fifteen CD patients and two ectopic ACTH syndrome (EAS) patients were studied. METHODS: Tumor fragments were submitted to RNA extraction, and GHSR-1a expression was studied through real-time qPCR and compared with normal tissue samples. The patients were also submitted to desmopressin test and vasopressin receptor type 1B (AVPR1B) mRNA analysis by qPCR. RESULTS: GHSR-1a expression was similar in normal pituitary samples and in corticotrophic tumor samples. GHSR-1a expression was higher in patients (CD and EAS) presenting in vivo response to GHRP-6. Higher expression of AVPR1B was observed in the EAS patients responsive to desmopressin, as well as in corticotrophic tumors, as compared with normal pituitary samples, but no correlation between AVPR1B expression and response to desmopressin was observed in the CD patients. CONCLUSIONS: Our results revealed a higher expression of GHSR-1a in the ACTH-dependent CS patients responsive to GHRP-6, suggesting an association between receptor gene expression and in vivo response to the secretagogue in both the CD and the EAS patients.

Serpin peptidase inhibitor clade A member 1 as a potential marker for malignancy in insulinomas.

PURPOSE: The biological behavior of insulinomas cannot be predicted based on histopathologic criteria in which the diagnosis of malignancy is confirmed by the presence of metastases. In this study, microarray and quantitative real-time reverse transcription-PCR were applied to identify differentially expressed genes between malignant and nonmalignant insulinomas to search for useful biomarkers to recognize the metastatic potential of insulinomas. EXPERIMENTAL DESIGN: Code Link human bioarrays were used to analyze differences in approximately 20,000 genes between six well-differentiated endocrine tumors of benign behavior compared with one well-differentiated endocrine carcinoma (WDEC) and three metastases of endocrine carcinomas (MEC). Quantitative real-time reverse transcription-PCR was used to validate differential expressions of five genes in a series of 35 sporadic insulinomas. Serpin peptidase inhibitor clade A member 1 (SERPINA1; alpha-1-antitrypsin) expression, identified as up-regulated in malignant insulinomas, was also evaluated by immunohistochemistry. RESULTS: Analysis of microarray data resulted in 230 differentially expressed genes. Gene Ontology analysis identified serine-type endopeptidase activity and serine-type endopeptidase inhibitor activity as pathways presenting significant differential expression. Protease serine 2 and complement factor B (from serine-type endopeptidase activity pathway) were respectively confirmed as up-regulated in well-differentiated endocrine tumors of benign behavior (WDET) and in WDEC/MEC. Angiotensinogen and SERPINA1 (from serine-type endopeptidase inhibitor activity pathway) were confirmed as up-regulated in WDEC/MEC. SERPINA1 was shown to be expressed in 85.7% of malignant versus 14.3% of nonmalignant insulinomas by immunohistochemistry. CONCLUSIONS: Our data are consistent to the possibility that SERPINA1 is a marker of malignancy in insulinomas. Given the widespread availability of antibody anti-alpha-1-antitrypsin in pathology services, SERPINA1 expression evaluation might be of clinical utility in recognizing patients more likely to develop an aggressive presentation.

Analysis of pancreatic adenocarcinoma tumor staging and resection according to previous body mass index and diabetes duration.

BACKGROUND/AIMS: Obesity and diabetes mellitus (DM) are associated with pancreatic cancer. The present study evaluated tumor staging and resection of pancreatic adenocarcinoma (PaC) according to previous Body Mass Index (BMI), BMI on admission and DM duration. METHODS: A retrospective analysis of 151 consecutive patients with PaC was performed: 73 were evaluated according to BMI preceding tumor-related weight loss and BMI on admission; 118 according to DM diagnosis; and 38 were assessed according to DM duration (less than 1 year [recent-onset] versus more than 2 years [long-standing]). RESULTS: There was no difference in the prevalence of tumor stage III or IV between previously normal weight and overweight/obese patients (56 vs. 42%, NS). Tumor resection rate was higher in previously obese than in previously lean patients (58 vs. 24%, p < 0.05). Tumor staging and resection were similar between normal weight and overweight/obese patients considering BMI on admission and diagnosis of DM. Weight loss was more pronounced in diabetic than in non-diabetic patients (21.7 vs. 13.3%, p < 0.01). Tumor staging and resection were similar between recent-onset and long-standing diabetic patients. CONCLUSION: Tumor resection rate was lower in previously normal weight patients. Diabetics lost more weight than non-diabetic patients. Neither BMI on admission nor the presence of DM nor DM duration influenced tumor staging or resection in PaC patients.

Modulation of hepatic microsomal triglyceride transfer protein (MTP) induced by S-nitroso-N-acetylcysteine in ob/ob mice.

We evaluated the effects of a potent NO donor, S-nitroso-N-acetylcysteine (SNAC), on microsomal triglyceride transfer protein (MTP) expression in ob/ob mice. NAFLD was induced in male ob/ob mice using a methionine-choline deficient diet (MCD) concomitantly with oral SNAC fed solution (n=5) or vehicle (control; n=5) by gavage daily for 4 weeks. Livers were collected for histology and for assessing MTP by RT-qPCR, Western blot, immunohistochemistry and immunogold electron microscopy analyses. Histological analysis showed diffuse macro and microvesicular steatosis, moderate hepatocellular ballooning and moderate inflammatory infiltrate in ob/ob mice fed the MCD diet. With SNAC, mice showed a marked reduction in liver steatosis (p<0.01), in parenchymal inflammation (p=0.02) and in MTP protein immunoexpression in zone III (p=0.05). Moreover, SNAC caused reduction of MTP protein in Western blot analysis (p<0.05). In contrast, MTP mRNA content was significantly higher (p<0.05) in mice receiving SNAC. Immuno-electron microscopy showed MTP localized in the rough endoplasmic reticulum of hepatocytes in both treated and untreated groups. However with SNAC treatment, MTP was also observed surrounding fat globules. Histological improvement mediated by a nitric oxide donor is associated with significantly altered expression and distribution of MTP in this animal model of fatty liver disease. Further studies are in progress to examine possible mechanisms and to develop SNAC as a possible therapy for human fatty liver disease.

PTTG expression in different experimental and human prolactinomas in relation to dopaminergic control of lactotropes.

BACKGROUND: Pituitary tumor transforming gene (pttg) is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Nevertheless, its expression in prolactinomas and its relation with the pituitary dopamine receptor 2 (D2R) are not well defined. We sought to determine the pituitary level of pttg in three different experimental models of prolactinomas with altered dopaminergic control of the pituitary: the dopaminergic D2R knockout female mouse, the estrogen-treated rat, and the senescent female rat. These three models shared the characteristics of increased pituitary weight, hyperprolactinemia, lactotrope hyperplasia and reduced or absent dopaminergic action at the pituitary level. We also studied samples from human macroprolactinomas, which were characterized as responsive or resistant to dopamine agonist therapy. RESULTS: When compared to female wild-type mice, pituitaries from female D2R knockout mice had decreased PTTG concentration, while no difference in pttg mRNA level was found. In senescent rats no difference in pituitary PTTG protein expression was found when compared to young rats. But, in young female rats treated with a synthetic estrogen (Diethylstylbestrol, 20 mg) PTTG protein expression was enhanced (P = 0.029). Therefore, in the three experimental models of prolactinomas, pituitary size was increased and there was hyperprolactinemia, but PTTG levels followed different patterns.Patients with macroprolactinomas were divided in those in which dopaminergic therapy normalized or failed to normalize prolactin levels (responsive and resistant, respectively). When pituitary pttg mRNA level was analyzed in these macroprolactinomas, no differences were found. We next analyzed estrogen action at the pituitary by measuring pituitary estrogen receptor alpha levels. The D2R knockout female mice have low estrogen levels and in accordance, pituitary estrogen receptors were increased (P = 0.047). On the other hand, in senescent rats estrogen levels were slightly though not significantly higher, and estrogen receptors were similar between groups. The estrogen-treated rats had high pharmacological levels of the synthetic estrogen, and estrogen receptors were markedly lower than in controls (P < 0.0001). Finally, in patients with dopamine resistant or responsive prolactinomas no significant differences in estrogen receptor alpha levels were found. Therefore, pituitary PTTG was increased only if estrogen action was increased, which correlated with a decrease in pituitary estrogen receptor level. CONCLUSION: We conclude that PTTG does not correlate with prolactin levels or tumor size in animal models of prolactinoma, and its pituitary content is not related to a decrease in dopaminergic control of the lactotrope, but may be influenced by estrogen action at the pituitary level. Therefore it is increased only in prolactinomas generated by estrogen treatment, and not in prolactinomas arising from deficient dopamine control, or in dopamine resistant compared with dopamine responsive human prolactinomas. These results are important in the search for reliable prognostic indicators for patients with pituitary adenomas which will make tumor-specific therapy possible, and help to elucidate the poorly understood phenomenon of pituitary tumorigenesis.

Hepatic gene expression profile associated with non-alcoholic steatohepatitis protection by S-nitroso-N-acetylcysteine in ob/ob mice.

BACKGROUND/AIMS: To understand the molecular mechanisms underlying non-alcoholic steatohepatitis (NASH) prevention by S-nitroso-N-acetylcysteine (SNAC), an NO donor that inhibits lipid peroxidation, we examined hepatic differentially expressed genes between ob/ob mice receiving or not SNAC treatment concomitantly with a methionine-choline deficient (MCD) diet. METHODS: Ob/ob mice were assigned to receive oral SNAC fed solution (MCD+SNAC group) or vehicle (MCD group) by gavage. After four weeks, histopathological analysis and microarray hybridizations were conducted in liver tissues from groups. GeneSifter system was used to identify differentially expressed genes and pathways according to Gene Ontology. RESULTS: NASH was absent in the MCD+SNAC group and no significant changes in food intake or body weight were observed in comparison to MCD group. After SNAC treatment, several genes belonging to oxidative phosphorylation, fatty acid biosynthesis, fatty acid metabolism and glutathione metabolism pathways were down-regulated in comparison to the MCD group. CONCLUSIONS: SNAC treatment promotes down regulation of several genes from fatty acid (FA) metabolism related pathways, possibly through abrogation of the cytotoxic effects of reactive oxygen species and lipid peroxides with consequent prevention of mitochondrial overload. Further studies are required to investigate the clinical implications of these findings, in attempt to develop novel therapeutic strategies for NAFLD treatment.