Biomedical subjects

Raghavendra G Mirmira

Publications and source records attributed to Raghavendra G Mirmira.

11 recordsLinked to original sources

PubMed2025

The Heterogeneity of Type 1 Diabetes: Implications for Pathogenesis, Prevention, and Treatment-2024 Diabetes, Diabetes Care, and Diabetologia Expert Forum.

This article summarizes the current understanding of the heterogeneity of type 1 diabetes from a June 2024 international Expert Forum organized by the editors of Diabetes, Diabetes Care, and Diabetologia. The Forum reviewed key factors contributing to the development and progression of type 1 diabetes and outlined specific, high-priority research questions. Knowledge gaps were identified, and, notably, opportunities to harness disease heterogeneity to develop personalized therapies were outlined. Herein, we summarize our discussions and review the heterogeneity of genetic risk and immunologic and metabolic phenotypes that influence and characterize type 1 diabetes progression (presented as a palette of risk factors). We discuss how these age-related factors determine disease aggressiveness (along gradients) and describe how variable immunogenetic pathways aggregate (into networks) to affect β-cell and other pancreatic pathologies to cause clinical disease at different ages and with variable severity (described as disease-related thresholds). Heterogeneity of pathogenesis and clinical severity opens avenues to prevention and intervention, including the potential of disease-modifying immunotherapy and islet cell replacement. We conclude with a call for 1) continued research to identify more factors contributing to the disease, both overall and in specific subgroups; 2) investigations focusing on both individuals who surpass metabolic and immune thresholds and develop diabetes and those who remain disease free with the same level of immunogenetic risk; and 3) efforts to identify where the current type 1 diabetes staging system may fall short and determine how it can be improved to capture and leverage heterogeneity in prevention and intervention strategies.

Humans↗

PubMed2025

A consensus guide to preclinical indirect calorimetry experiments.

Understanding the complex factors influencing mammalian metabolism and body weight homeostasis is a long-standing challenge requiring knowledge of energy intake, absorption and expenditure. Using measurements of respiratory gas exchange, indirect calorimetry can provide non-invasive estimates of whole-body energy expenditure. However, inconsistent measurement units and flawed data normalization methods have slowed progress in this field. This guide aims to establish consensus standards to unify indirect calorimetry experiments and their analysis for more consistent, meaningful and reproducible results. By establishing community-driven standards, we hope to facilitate data comparison across research datasets. This advance will allow the creation of an in-depth, machine-readable data repository built on shared standards. This overdue initiative stands to markedly improve the accuracy and depth of efforts to interrogate mammalian metabolism. Data sharing according to established best practices will also accelerate the translation of basic findings into clinical applications for metabolic diseases afflicting global populations.

Calorimetry, Indirect↗

PubMed2006

Role of chromatin accessibility in the occupancy and transcription of the insulin gene by the pancreatic and duodenal homeobox factor 1.

The pancreatic and duodenal homeobox factor 1 (Pdx-1) is a Hox-like transcription factor that is responsible for the activation of the insulin gene. Previous studies have demonstrated the interaction in vitro of Pdx-1 with short (20-40 nucleotide) DNA fragments corresponding to A boxes of the insulin promoter. Precisely how Pdx-1 binds to DNA in the complex milieu of chromatin, however, has never been studied. In this study, we explored how Pdx-1-DNA interactions might be influenced by chromatin accessibility at the insulin gene in beta-cells (betaTC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in betaTC3 cells but not in mPAC cells, a finding that is independent of the intracellular Pdx-1 protein concentration. Based on micrococcal nuclease protection assays, the difference in promoter binding between the two cell types appears to be secondary to chromatin accessibility at predicted Pdx-1 binding sites between bp -126 to -296 (relative to the transcriptional start site) of the insulin promoter. Binding studies using purified Pdx-1 and reconstituted chromatin in vitro suggest that the positioning of a nucleosome(s) within this crucial region of the promoter might account for differences in chromatin accessibility. Consistent with these observations, fluorescence colocalization studies show that Pdx-1 does not occupy regions of compacted, nucleosome-rich chromatin within the nucleus. Our findings suggest a model whereby insulin transcription in the beta-cell is at least partially facilitated by enhanced chromatin accessibility within a crucial regulatory region between bp -126 to -296, thereby permitting occupancy by transactivators such as Pdx-1.

Acetylation↗

PubMed2005

Pdx-1 links histone H3-Lys-4 methylation to RNA polymerase II elongation during activation of insulin transcription.

Expression of the insulin gene is nearly exclusive to the beta cells of the pancreatic islets. Although the sequence-specific transcription factors that regulate insulin expression have been well studied, the interrelationship between these factors, chromatin structure, and transcriptional elongation by RNA polymerase II (pol II) has remained undefined. In this regard, recent studies have begun to establish a role for the methylation of histone H3 in the initiation or elongation of transcription by pol II. To determine a role for the transcriptional activator Pdx-1 in the maintenance of chromatin structure and pol II recruitment at the insulin gene, we performed small interfering RNA-mediated knockdown of Pdx-1 in betaTC3 cells and subsequently studied histone modifications and pol II recruitment by chromatin immunoprecipitation. We demonstrated here that the 50% fall in insulin transcription following knockdown of Pdx-1 is accompanied by a 60% fall in dimethylated histone H3-Lys-4 at the insulin promoter. H3-Lys-4 methylation at the insulin promoter may be mediated, at least partially, by the methyltransferase Set9. Immunohistochemical analysis revealed that Set9 is expressed in an islet-enriched pattern in the pancreas, similar to the pattern of Pdx-1 expression. The recruitment of Set9 to the insulin gene appears to be a consequence of its direct interaction with Pdx-1, and small interfering RNA-mediated knockdown of Set9 attenuates insulin transcription. Pdx-1 knockdown was also associated with an overall shift in the recruitment of pol II isoforms to the insulin gene, from an elongation isoform (Ser(P)-2) to an initiation isoform (Ser(P)-5). Our findings therefore suggest a model whereby Pdx-1 plays a novel role in linking H3-Lys-4 dimethylation and pol II elongation to insulin transcription.

Adenoviridae↗

PubMed2005

The C-terminal domain of the beta cell homeodomain factor Nkx6.1 enhances sequence-selective DNA binding at the insulin promoter.

The Hox-like factor Nkx6.1 is required for the formation and secretory function of insulin-producing beta cells, and has the capacity to activate or repress the transcription of relevant target genes in a DNA-context dependent manner. A key determinant of transcriptional activity by Nkx6.1 may be its C terminus, which has been suggested to interfere with DNA binding. To determine how the C terminus modulates homeodomain binding, we assessed the nature of Nkx6.1-DNA interactions at the insulin promoter. By quantitative gel shift analysis, we demonstrate that although the C terminus of Nkx6.1 mitigates the affinity of the homeodomain for DNA slightly (about 2-fold), it enhances the selectivity of the homeodomain for TAAT DNA sequences nearly 10-fold. By reporter gene analysis, this selectivity is also functionally preserved in mammalian cells in vivo. Based on deletional and mutational studies, the sequence selectivity imparted by the C terminus appears to be mediated by a stretch of highly conserved residues between amino acids 318 and 338. Strikingly, these residues impart minimal changes to the secondary structure of the unbound protein as assessed by circular dichroism spectroscopy, suggesting that conformational adjustments of the homeodomain that occur upon binding to DNA may play a more important role in sequence selectivity. The C terminus of Nkx6.1 also functions in a modular fashion, as it can confer similar DNA binding properties when fused to the heterologous homeodomain of Pdx-1. Taken together, our data suggest a model whereby the Nkx6.1 C terminus may function in a regulatory manner by imposing specific functional constraints upon the protein. These constraints may serve to modulate the potential of Nkx6.1 to both recognize target genes and regulate their transcription.

Animals↗

PubMed2005

The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet beta cells.

We have previously described rat insulinoma INS-1-derived cell lines with robust or poor glucose-stimulated insulin secretion (GSIS). In the current study, we have further resolved these lines into three classes: class 1, glucose-unresponsive/glucagon-expressing; class 2, glucose-unresponsive/glucagon-negative; and class 3, glucose-responsive/glucagon-negative. The transcription factor Nkx2.2 was expressed with relative abundance of 3.3, 1.0, and 1.0 in class 1, class 2, and class 3 cells, respectively, whereas Nkx6.1 expression had the opposite trend: 1.0, 2.6, and 6.4 in class 1, class 2, and class 3 cells, respectively. In class 1 cells, overexpressed Nkx6.1 suppressed glucagon expression but did not affect the levels of several other prominent beta cell transcription factors. RNA interference (RNAi)-mediated suppression of Nkx6.1 in class 3 cells resulted in a doubling of glucagon mRNA, with no effect on Pdx1 levels, whereas suppression of Pdx1 in class 3 cells caused a 12-fold increase in glucagon transcript levels, demonstrating independent effects of Nkx6.1 and Pdx1 on glucagon expression in beta cell lines. RNAi-mediated suppression of Nkx6.1 expression in class 3 cells also caused a decrease in GSIS from 13.9- to 3.7-fold, whereas suppression of Pdx1 reduced absolute amounts of insulin secretion without affecting fold response. Finally, RNAi-mediated suppression of Nkx6.1 mRNA in primary rat islets was accompanied by a significant decrease in GSIS relative to control cells. In sum, our studies have revealed roles for Nkx6.1 in suppression of glucagon expression and control of GSIS in islet beta cells.

Adenoviridae↗

PubMed2005

Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes.

The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.

Adenoviridae↗

PubMed2004

The transcriptional repressor Nkx6.1 also functions as a deoxyribonucleic acid context-dependent transcriptional activator during pancreatic beta-cell differentiation: evidence for feedback activation of the nkx6.1 gene by Nkx6.1.

In the pancreas, the NK homeodomain transcription factor Nkx6.1 is required for the development of beta-cells and is believed to function as a potent repressor of transcription upon binding to A/T-rich sequences within the promoter region of target genes. Because the nkx6.1 promoter itself contains several such sequences, we considered the possibility that the expression level and restricted pattern of the nkx6.1 gene might be precisely regulated by one or more homeodomain transcription factors, including Nkx6.1 itself. In this report, we identify a novel beta-cell-specific enhancer element in the nkx6.1 gene between -157 and -30 bp (relative to the transcriptional start site) that harbors a conserved A/T-containing sequence flanked by G/C-rich stretches. Although the islet homeodomain-containing activator Pdx-1 was unable to stimulate transcription of a reporter gene through this enhancer element in mammalian cell lines, strikingly, Nkx6.1 robustly activated transcription through direct interaction with the A/T-rich sequence in this element. We demonstrate that this activation is indeed transcriptional in nature (and not secondary to translational effects) and is mediated by a modular acidic sequence within the COOH-terminal domain of Nkx6.1. We show by EMSAs that Nkx6.1 binds to the beta-cell-specific enhancer in vitro and by chromatin immunoprecipitation assays that Nkx6.1 natively occupies this region in vivo in betaTC3 cells. We therefore conclude that Nkx6.1 is a bifunctional transcription factor that serves to maintain the specific expression of its own gene during beta-cell differentiation while simultaneously effecting broader gene repression events.

Animals↗

PubMed2003

Covalent histone modifications underlie the developmental regulation of insulin gene transcription in pancreatic beta cells.

Histone modifying enzymes contribute to the activation or inactivation of transcription by ultimately catalyzing the unfolding or further compaction, respectively, of chromatin structure. Actively transcribed genes are typically hyperacetylated at Lys residues of histones H3 and H4 and hypermethylated at Lys-4 of histone H3 (H3-K4). To determine whether covalent histone modifications play a role in the beta cell-specific expression of the insulin gene, we performed chromatin immunoprecipitation assays using anti-histone antibodies and extracts from beta cell lines, non-beta cell lines, and ES cells, and quantitated specific histone modifications at the insulin promoter by real-time PCR. Our studies reveal that the proximal insulin promoter is hyperacetylated at histone H3 only in beta cells. This hyperacetylation is highly correlated to recruitment of the histone acetyltransferase p300 to the proximal promoter in beta cells, and is consistent with the role of hyperacetylation in promoting euchromatin formation. We also observed that the proximal insulin promoter of beta cells is hypermethylated at H3-K4, and that this modification is correlated to the recruitment of the histone methyltransferase SET7/9 to the promoter. ES cells demonstrate a histone modification pattern intermediate between that of beta cells and non-beta cells, and is consistent with their potential to express the insulin gene. We therefore propose a model in which insulin transcription in the beta cell is facilitated by a unique combination of transcription factors that acts in the setting of an open, euchromatic structure of the insulin gene.

Acetylation↗

PubMed2003

Transcription factors direct the development and function of pancreatic beta cells.

Transcription factors orchestrate intricate pathways of cellular growth and differentiation by regulating the rate of transcription of an array of genes. Genetic and biochemical studies have begun to unravel the complex cascade of factors that controls the proliferation and differentiation of cells in the developing pancreas. The specific pathway leading to the development of the insulin-secreting beta cell has been a focus of many of these studies because an understanding of the transcription factors governing this pathway will be crucial to the engineering of new beta cells to cure diabetes. In recent years, the number of transcription factors that has been implicated in beta-cell differentiation and function has grown considerably. Here, we outline the known role of transcription factors in beta-cell development, and describe how these factors form a network of gene activation signals that mediates insulin transcription.

Animals↗

PubMed2002

Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1. Importance of chromatin structure in directing promoter binding.

The transcription factor Pdx1 is expressed in the pancreatic beta-cell, where it is believed to regulate several beta-cell-specific genes. Whereas binding by Pdx1 to elements of beta-cell genes has been demonstrated in vitro, almost none of these genes has been demonstrated to be a direct binding target for Pdx1 within cells (where complex chromatin structure exists). To determine which beta-cell promoters are bound by Pdx1 in vivo, we performed chromatin immunoprecipitation assays using Pdx1 antiserum and chromatin from beta-TC3 cells and Pdx1-transfected NIH3T3 cells and subsequently quantitated co-immunoprecipitated promoters using real-time PCR. We compared these in vivo findings to parallel immunoprecipitations in which Pdx1 was allowed to bind to promoter fragments in in vitro reactions. Our results show that in all cells Pdx1 binds strongly to the insulin, islet amyloid polypeptide, glucagon, Pdx1, and Pax4 promoters, whereas it does not bind to either the glucose transporter type 2 or albumin promoters. In addition, no binding by Pdx1 to the glucokinase promoter was observed in beta-cells. In contrast, in in vitro immunoprecipitations, Pdx1 bound all promoters to an extent approximately proportional to the number of Pdx1 binding sites. Our findings suggest a critical role for chromatin structure in directing the promoter binding selectivity of Pdx1 in beta-cells and non-beta-cells.

Animals↗