PubMed Health⌕ Search

Biomedical subjects

Elisabetta Manduchi

Publications and source records attributed to Elisabetta Manduchi.

4 recordsLinked to original sources
PubMed2025

G6PC2 controls glucagon secretion by defining the set point for glucose in pancreatic α cells.

Elevated glucagon concentrations have been reported in patients with type 2 diabetes (T2D). A critical role for α cell-intrinsic mechanisms in regulating glucagon secretion was previously established through genetic manipulation of the glycolytic enzyme glucokinase (GCK) in mice. Genetic variation at the glucose-6-phosphatase catalytic subunit 2 (G6PC2) locus, encoding an enzyme that opposes GCK, has been reproducibly associated with fasting blood glucose and hemoglobin A1c. Here, we found that trait-associated variants in the G6PC2 promoter are located in open chromatin not just in β but also in α cells and documented allele-specific G6PC2 expression of linked variants in human α cells. Using α cell-specific gene ablation of G6pc2 in mice, we showed that this gene plays a critical role in controlling glucose suppression of amino acid-stimulated glucagon secretion independent of alterations in insulin output, islet hormone content, or islet morphology, findings that we confirmed in primary human α cells. Collectively, our data demonstrate that G6PC2 affects glycemic control via its action in α cells and possibly suggest that G6PC2 inhibitors might help control blood glucose through a bihormonal mechanism.

Glucagon↗
PubMed2003

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA.

Although mRNA amplification is necessary for microarray analyses from limited amounts of cells and tissues, the accuracy of transcription profiles following amplification has not been well characterized. We tested the fidelity of differential gene expression following linear amplification by T7-mediated transcription in a well-established in vitro model of cytokine [tumor necrosis factor alpha (TNFalpha)]-stimulated human endothelial cells using filter arrays of 13,824 human cDNAs. Transcriptional profiles generated from amplified antisense RNA (aRNA) (from 100 ng total RNA, approximately 1 ng mRNA) were compared with profiles generated from unamplified RNA originating from the same homogeneous pool. Amplification accurately identified TNFalpha-induced differential expression in 94% of the genes detected using unamplified samples. Furthermore, an additional 1,150 genes were identified as putatively differentially expressed using amplified RNA which remained undetected using unamplified RNA. Of genes sampled from this set, 67% were validated by quantitative real-time PCR as truly differentially expressed. Thus, in addition to demonstrating fidelity in gene expression relative to unamplified samples, linear amplification results in improved sensitivity of detection and enhances the discovery potential of high-throughput screening by microarrays.

Bias↗
PubMed2003

Transcriptional program of the endocrine pancreas in mice and humans.

The Endocrine Pancreas Consortium was formed in late 1999 to derive and sequence cDNA libraries enriched for rare transcripts expressed in the mammalian endocrine pancreas. Over the past 3 years, the Consortium has generated 20 cDNA libraries from mouse and human pancreatic tissues and deposited >150,000 sequences into the public expressed sequence tag databases. A special effort was made to enrich for cDNAs from the endocrine pancreas by constructing libraries from isolated islets. In addition, we constructed a library in which fetal pancreas from Neurogenin 3 null mice, which consists of only exocrine and duct cells, was subtracted from fetal wild-type pancreas to enrich for the transcripts from the endocrine compartment. Sequence analysis showed that these clones cluster into 9,464 assembly groups (approximating unique transcripts) for the mouse and 13,910 for the human sequences. Of these, >4,300 were unique to Consortium libraries. We have assembled a core clone set containing one cDNA for each assembly group for the mouse and have constructed the corresponding microarray, termed "PancChip 4.0," which contains >9,000 nonredundant elements. We show that this PancChip is highly enriched for genes expressed in the endocrine pancreas. The mouse and human clone sets and corresponding arrays will be important resources for diabetes research.

Animals↗
PubMed2002

Comparison of different labeling methods for two-channel high-density microarray experiments.

In this report we evaluate three methods for labeling nucleic acids to be hybridized to a cDNA microarray: direct labeling, indirect amino-allyl labeling, and the dendrimer labeling method (Genisphere). The dendrimer method requires the smallest quantity of sample, 2.5 microg of total RNA compared with 20 microg with the direct or indirect methods. Therefore, we wanted to know whether the performance of the dendrimer method is comparable to the other methods, or whether significant information is lost. Performance can be considered in terms of sensitivity, dynamic range, and reproducibility of the quantitative signals for gene intensity. We compared the three labeling methods by generating three sets of eight self-to-self hybridizations using the same total RNA sample in all cases ("replicate study"). In our analysis, we controlled for the effects of print-tip and background subtraction biases. We also performed a smaller study, namely, a dilution series study with five dilution points per labeling method, to evaluate one aspect of predictive ability. From the replicate study, the dendrimer method appeared to perform as well, and often better, with respect to reproducibility and ability to detect expression. However, in the dilution series study, this method was outperformed by the other two in terms of predictive ability and did not perform very well. These findings are helping to guide our decisions on what labeling method to use for subsequent studies, based on the purpose of a specific study and its limitations in terms of available material.

Fluorescent Dyes↗