Multiple promoter and enhancer differences likely contribute to augmented G6PC2 expression in human versus mouse pancreatic islet alpha cells.

G6PC2 encodes a glucose-6-phosphatase catalytic subunit that opposes the action of glucokinase in pancreatic islets, thereby modulating the sensitivity of insulin and glucagon secretion to glucose. In mice, G6pc2 is expressed at ~20-fold higher levels in ß-cells than in α-cells, whereas in humans G6PC2 is expressed at only ~5-fold higher levels in ß-cells. We therefore hypothesize that G6PC2 likely influences glucagon secretion to a greater degree in humans. With a view to generating a humanized mouse that recapitulates augmented G6PC2 expression levels in α-cells, we sought to identify the genomic regions that confer differential mouse G6pc2 expression in α-cells versus ß-cells as well as the evolutionary changes that have altered this ratio in humans. Studies in islet-derived cell lines suggest that the elevated G6pc2 expression in mouse ß-cells versus α-cells is mainly due to a difference in the relative activity of the proximal G6pc2 promoter in these cell types. Similarly, the smaller difference in G6PC2 expression between α-cells and ß-cells in humans is potentially explained by a change in relative proximal G6PC2 promoter activity. However, we show that both glucocorticoid levels and multiple differences in the relative activity of eight transcriptional enhancers between mice and humans likely contribute to differential G6PC2 expression. Finally, we show that a mouse-specific non-coding RNA, Gm13613, whose expression is controlled by G6pc2 enhancer I, does not regulate G6pc2 expression, indicating that altered expression of Gm13613 in a humanized mouse that contains both the human promoter and enhancers should not affect G6PC2 function.

A novel dual balloon rectal catheter for use in the postoperative assessment of low rectal anastomoses following low anterior resection.

Following low anterior resection (LAR) of the colon, an image-guided assessment of the anastomosis for leak is typically performed using an enema via a rectal catheter, whether by CT or fluoroscopy. However, there is potential for poor assessment due to inappropriate catheter positioning as well as potential risk that the anastomosis becomes compromised by the balloon inflation. This article discusses the adaptation of a novel double-balloon catheter (originally designed by a member of our institution for use in pediatric intussusception reduction) for assessment of low rectal anastomoses. The goal of this technical note is to demonstrate our experience with this catheter, primarily through example cases, and explain its potential for optimizing colon distension, minimizing improper catheter placement, and potentially reducing the risk of iatrogenic anastomosis disruption.

Multi-Center Follow-up Study to Develop a Classification System Which Differentiates Mucinous Cystic Neoplasm of the Liver and Benign Hepatic Cyst Using Machine Learning.

RATIONALE AND OBJECTIVES: To date, no clinically useful classification system has been developed for reliably differentiating mucinous cystic neoplasm (MCN) from a benign hepatic cyst (BHC) in the liver. The objective was to use machine learning and a multi-center study design to develop and assess the performance of a novel classification system for predicting whether a hepatic cystic lesion represents MCN or BHC. MATERIALS AND METHODS: A multi-center cohort study identified 154 surgically resected hepatic cystic lesions in 154 subjects which were pathologic confirmed as MCN (43) or BHC (111). Readers at each institution recorded seven pre-determined imaging features previously identified as potential differentiating features from prior publications. The contribution of each of these features to differentiating MCN from BHC was assessed by machine learning to develop an optimal classification system. RESULTS: Although several of the assessed imaging features demonstrated statistical significance, only 3 imaging features were found by machine learning to significantly contribute to a potential classification system: (1) solid enhancing nodule (2) all septations arising from an external macro-lobulation (3) whether the lesion was solitary or one of multiple cystic liver lesions. The optimal classification system had only four categories and correctly identified 144/154 lesion (93.5%). CONCLUSION: This multi-center follow-up study was able to use machine learning to develop a highly accurate classification system for differentiation of hepatic MCN from BHC, which could be readily applied to clinical practice.

Non-contrast and portal venous phase computed tomography in breast cancer hepatic metastases: comparison of tumor measurements and impact on response assessment.

BACKGROUND: For many common malignancies, including breast cancer, evaluation for metastatic disease using multiphase computed tomography (CT) has fallen out of favor and been replaced by studies performed only in the portal venous phase. However, differences in tumor vascularity could produce differences in appearance on post-contrast imaging. PURPOSE: To assess non-contrast phase and portal venous phase computed tomography in detection and measurement of hepatic metastases from breast carcinoma. MATERIALS AND METHODS: A total of 75 CT scans from 52 breast cancer patients were independently assessed by three body imagers for lesion presence, number and size. Readers randomly assessed portal venous phase or combined phase images at one session with cross-over reads performed four to six weeks later. RESULTS: In the 58% of cases where index lesions measured larger on combined phase, the mean difference in lesion size was 5.7 mm. In this group, combined phase reads demonstrated an 8.4 mm increase in sum of largest diameters, and a mean percentage sum of largest diameters increase of 19% compared to portal venous phase-only reads. CONCLUSION: Addition of non-contrast phase images results in increased index lesion size in most patients with hepatic metastases from breast cancer. If only the portal venous phase is utilized, there is potential for incorrectly diagnosing disease progression on follow-up due to underestimation of lesion size.

Added value of iodine-specific imaging and virtual non-contrast imaging for gastrointestinal assessment using dual-energy computed tomography.

Dual-energy computed tomography (DECT) has become increasingly available and can be readily incorporated into clinical practice. Although DECT can provide a wide variety of spectral imaging reconstructions, most clinically valuable information is available from a limited number of standard image reconstructions including virtual non-contrast and iodine overlay. The combination of these standard reconstructions can be used for specific diagnostic tasks that provide added value over traditional CT protocols. In this pictorial essay, the added value of these standard reconstructed images will be demonstrated by case examples for diseases specifically related to the gastrointestinal system.

Combined Qualitative and Quantitative Assessment of Low-Attenuation Renal Lesions Improves Identification of Renal Malignancy on Noncontrast Computed Tomography.

OBJECTIVE: The objective of this study was to assess renal lesions measuring less than 20 Hounsfield units (HU) on noncontrast computed tomography (NCT). METHODS: Twenty-one (18.1%) of 116 consecutive pathologically proven renal cell carcinomas measured less than 20 HU on NCT and were compared with 40 confirmed benign cysts also measuring less than 20 HU. All lesions were assessed qualitatively (heterogeneous or homogenous) by 3 blinded readers and quantitatively with commercially available textural analysis software. Finally, a combined assessment was performed. RESULTS: Qualitative assessment performed well (sensitivity, 76%-90%; specificity, 70%-88%). Quantitative assessment revealed mean positive pixels as having the highest performance (area under the curve, 0.912; sensitivity, 90%; specificity, 80% at a cutoff value of 21). The combined assessment, using the mean positive pixel cutoff, improved the sensitivity (reader 1, 100%; reader 2, 95%; and reader 3, 95%). CONCLUSION: Qualitative and quantitative assessments have relatively good performance, but the combination can nearly eliminate renal cell carcinomas being missed on NCT.

Limitation of Virtual Noncontrasted Images in Evaluation of a Liver Lesion Status Post Transarterial Chemoembolization.

The authors describe a case of a patient with a solitary hepatocellular carcinoma status post transarterial chemoembolization. Follow-up imaging was performed using dual-energy computed tomography. The study was performed with and without contrast and a virtual noncontrast data set was constructed from the postcontrast images. The evaluation of this patient status post transarterial chemoembolization with virtual noncontrast alone erroneously suggested enhancement and viable tumor. However, examination of true noncontrast images revealed these findings to be due to the subtraction of iodine in Ethiodol within the treated lesion.

Genetic and functional assessment of the role of the rs13431652-A and rs573225-A alleles in the G6PC2 promoter that are strongly associated with elevated fasting glucose levels.

OBJECTIVE: Genome-wide association studies have identified a single nucleotide polymorphism (SNP), rs560887, located in a G6PC2 intron that is highly correlated with variations in fasting plasma glucose (FPG). G6PC2 encodes an islet-specific glucose-6-phosphatase catalytic subunit. This study examines the contribution of two G6PC2 promoter SNPs, rs13431652 and rs573225, to the association signal. RESEARCH DESIGN AND METHODS: We genotyped 9,532 normal FPG participants (FPG <6.1 mmol/l) for three G6PC2 SNPs, rs13431652 (distal promoter), rs573225 (proximal promoter), rs560887 (3rd intron). We used regression analyses adjusted for age, sex, and BMI to assess the association with FPG and haplotype analyses to assess comparative SNP contributions. Fusion gene and gel retardation analyses characterized the effect of rs13431652 and rs573225 on G6PC2 promoter activity and transcription factor binding. RESULTS: Genetic analyses provide evidence for a strong contribution of the promoter SNPs to FPG variability at the G6PC2 locus (rs13431652: ß = 0.075, P = 3.6 × 10(-35); rs573225 ß = 0.073 P = 3.6 × 10(-34)), in addition to rs560887 (ß = 0.071, P = 1.2 × 10(-31)). The rs13431652-A and rs573225-A alleles promote increased NF-Y and Foxa2 binding, respectively. The rs13431652-A allele is associated with increased FPG and elevated promoter activity, consistent with the function of G6PC2 in pancreatic islets. In contrast, the rs573225-A allele is associated with elevated FPG but reduced promoter activity. CONCLUSIONS: Genetic and in situ functional data support a potential role for rs13431652, but not rs573225, as a causative SNP linking G6PC2 to variations in FPG, though a causative role for rs573225 in vivo cannot be ruled out.

Insulin and epidermal growth factor suppress basal glucose-6-phosphatase catalytic subunit gene transcription through overlapping but distinct mechanisms.

The G6Pase (glucose-6-phosphatase catalytic subunit) catalyses the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate to glucose. We show here that, in HepG2 hepatoma cells, EGF (epidermal growth factor) inhibits basal mouse G6Pase fusion gene transcription. Several studies have shown that insulin represses basal mouse G6Pase fusion gene transcription through FOXO1 (forkhead box O1), but Stoffel and colleagues have recently suggested that insulin can also regulate gene transcription through FOXA2 (forkhead box A2) [Wolfrum, Asilmaz, Luca, Friedman and Stoffel (2003) Proc. Natl. Acad. Sci. 100, 11624-11629]. A combined GR (glucocorticoid receptor)-FOXA2 binding site is located between -185 and -174 in the mouse G6Pase promoter overlapping two FOXO1 binding sites located between (-188 and -182) and (-174 and -168). Selective mutation of the FOXO1 binding sites reduced the effect of insulin, whereas mutation of the GR/FOXA2 binding site had no effect on the insulin response. In contrast, selective mutation of the FOXO1 and GR/FOXA2 binding sites both reduced the effect of EGF. The effect of these mutations was additive, since the combined mutation of both FOXO1 and GR/FOXA2 binding sites reduced the effect of EGF to a greater extent than the individual mutations. These results suggest that, in HepG2 cells, GR and/or FOXA2 are required for the inhibition of basal G6Pase gene transcription by EGF but not insulin. EGF also inhibits hepatic G6Pase gene expression in vivo, but in cultured hepatocytes EGF has the opposite effect of stimulating expression, an observation that may be explained by a switch in ErbB receptor sub-type expression following hepatocyte isolation.

Foxa2 and MafA regulate islet-specific glucose-6-phosphatase catalytic subunit-related protein gene expression.

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP/G6PC2) is a major autoantigen in both mouse and human type 1 diabetes. IGRP is selectively expressed in islet beta cells and polymorphisms in the IGRP gene have recently been associated with variations in fasting blood glucose levels and cardiovascular-associated mortality in humans. Chromatin immunoprecipitation (ChIP) assays have shown that the IGRP promoter binds the islet-enriched transcription factors Pax-6 and BETA2. We show here, again using ChIP assays, that the IGRP promoter also binds the islet-enriched transcription factors MafA and Foxa2. Single binding sites for these factors were identified in the proximal IGRP promoter, mutation of which resulted in decreased IGRP fusion gene expression in betaTC-3, Hamster insulinoma tumor (HIT), and Min6 cells. ChiP assays have shown that the islet-enriched transcription factor Pdx-1 also binds the IGRP promoter, but mutational analysis of four Pdx-1 binding sites in the proximal IGRP promoter revealed surprisingly little effect of Pdx-1 binding on IGRP fusion gene expression in betaTC-3 cells. In contrast, in both HIT and Min6 cells mutation of these four Pdx-1 binding sites resulted in a approximately 50% reduction in fusion gene expression. These data suggest that the same group of islet-enriched transcription factors, namely Pdx-1, Pax-6, MafA, BETA2, and Foxa2, directly or indirectly regulate expression of the two major autoantigens in type 1 diabetes.

Long-range enhancers are required to maintain expression of the autoantigen islet-specific glucose-6-phosphatase catalytic subunit-related protein in adult mouse islets in vivo.

OBJECTIVE: Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet beta-cells and is a major autoantigen in both mouse and human type 1 diabetes. This study describes the use of a combination of transgenic and transfection approaches to characterize the gene regions that confer the islet-specific expression of IGRP. RESEARCH DESIGN AND METHODS: Transgenic mice were generated containing the IGRP promoter sequence from -306, -911, or -3911 to +3 ligated to a LacZ reporter gene. Transgene expression was monitored by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of pancreatic tissue. RESULTS: In all the transgenic mice, robust LacZ expression was detected in newborn mouse islets, but expression became mosaic as animals aged, suggesting that additional elements are required for the maintenance of IGRP gene expression. VISTA analyses identified two conserved regions in the distal IGRP promoter and one in the third intron. Transfection experiments demonstrated that all three regions confer enhanced luciferase reporter gene expression in beta TC-3 cells when ligated to a minimal IGRP promoter. A transgene containing all three conserved regions was generated by using a bacterial recombination strategy to insert a LacZ cassette into exon 5 of the IGRP gene. Transgenic mice containing a 15-kbp fragment of the IGRP gene were then generated. This transgene conferred LacZ expression in newborn mouse islets; however, expression was still suppressed as animals aged. CONCLUSIONS: The data suggest that long-range enhancers 5' or 3' of the IGRP gene are required for the maintenance of IGRP gene expression in adult mice.

Gluconeogenesis: re-evaluating the FOXO1-PGC-1alpha connection.

Increased expression of the gene encoding the enzyme glucose-6-phosphatase (G6Pase) contributes to the increased production of glucose by the liver that occurs in individuals with diabetes. Puigserver et al. show that the transcription factor FOXO1 and the transcriptional co-activator PGC-1alpha act synergistically to stimulate the expression of genes in the gluconeogenesis pathway and propose that PGC-1alpha acts, in part, directly through FOXO1. Here we show that FOXO1 is neither required nor sufficient for the stimulation of G6Pase-luciferase fusion gene expression by PGC-1alpha. Our results indicate that the transcriptional interaction between FOXO1 and PGC-1alpha is indirect.