Browsing by Author "Kanojia, Sukrati"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    The Chd4 subunit of the NuRD complex regulates Pdx1-controlled genes involved in β-cell function
    (Bioscientifica, 2022-06-14) Davidson, Rebecca K.; Weaver, Staci A.; Casey, Nolan; Kanojia, Sukrati; Hogarth, Elise; Schneider Aguirre, Rebecca; Sims, Emily K.; Evans-Molina, Carmella; Spaeth, Jason M.; Biochemistry and Molecular Biology, School of Medicine
    Type 2 diabetes (T2D) is associated with loss of transcription factors (TFs) from a subset of failing β-cells. Among these TFs is Pdx1, which controls the expression of numerous genes involved in maintaining β-cell function and identity. Pdx1 activity is modulated by transcriptional coregulators and has recently been shown, through an unbiased screen, to interact with the Chd4 ATPase subunit of the nucleosome remodeling and deacetylase complex. Chd4 contributes to the maintenance of cellular identity and functional status of numerous different cell types. Here, we demonstrated that Pdx1 dynamically interacts with Chd4 under physiological and stimulatory conditions within islet β-cells and established a fundamental role for Chd4 in regulating insulin secretion and modulating numerous Pdx1-bound genes in vitro, including the MafA TF, where we discovered Chd4 is bound to the MafA region 3 enhancer. Furthermore, we found that Pdx1:Chd4 interactions are significantly compromised in islet β-cells under metabolically induced stress in vivo and in human donor tissues with T2D. Our findings establish a fundamental role for Chd4 in regulating insulin secretion and modulating Pdx1-bound genes in vitro, and disruption of Pdx1:Chd4 interactions coincides with β-cell dysfunction associated with T2D.
  • Thumbnail Image
    Item
    Cigarette smoke exposure impairs β-cell function through activation of oxidative stress and ceramide accumulation
    (Elsevier, 2020-07) Tong, Xin; Chaudhry, Zunaira; Lee, Chih-Chun; Bone, Robert N.; Kanojia, Sukrati; Maddatu, Judith; Sohn, Paul; Weaver, Staci A.; Robertson, Morgan A.; Petrache, Irina; Evans-Molina, Carmella; Kono, Tatsuyoshi; Medicine, School of Medicine
    Objectives Epidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic β-cell function using rodent and in vitro models. Methods Beginning at 8 weeks of age, C57BL/6 J mice were concurrently fed a high-fat diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured INS-1 β-cells and primary islets were exposed ex vivo to CS extract (CSE), and β-cell function and viability were tested. Since CS increases ceramide accumulation in the lung and these bioactive sphingolipids have been implicated in pancreatic β-cell dysfunction in diabetes, islet and β-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry. Results Compared to HFD-fed, ambient air-exposed mice, HFD-fed and CS-exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and had accelerated worsening of their glucose tolerance. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of β-cell dysfunction, significantly lower β-cell mass (p = 0.017), reduced β-cell proliferation (p = 0.006), and increased islet ceramide content compared to non-smoking control mice. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide levels, which was correlated with reduced insulin gene expression and glucose-stimulated insulin secretion, and increased β-cell oxidative and endoplasmic reticulum (ER) stress. Treatment with the antioxidant N-acetylcysteine markedly attenuated the effects of CSE on ceramide levels, restored β-cell function and survival, and increased cyclin D2 expression, while also reducing activation of β-cell ER and oxidative stress. Conclusions Our results indicate that CS exposure leads to impaired insulin production, processing, secretion and reduced β-cell viability and proliferation. These effects were linked to increased β-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of the mechanisms by which CS exposure impairs β-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.
  • Thumbnail Image
    Item
    The Contribution of Transcriptional Coregulators in the Maintenance of β-cell Function and Identity
    (Endocrine Society, 2021) Davidson, Rebecca K.; Kanojia, Sukrati; Spaeth, Jason M.; Biochemistry and Molecular Biology, School of Medicine
    Islet β-cell dysfunction that leads to impaired insulin secretion is a principal source of pathology of diabetes. In type 2 diabetes, this breakdown in β-cell health is associated with compromised islet-enriched transcription factor (TF) activity that disrupts gene expression programs essential for cell function and identity. TF activity is modulated by recruited coregulators that govern activation and/or repression of target gene expression, thereby providing a supporting layer of control. To date, more than 350 coregulators have been discovered that coordinate nucleosome rearrangements, modify histones, and physically bridge general transcriptional machinery to recruited TFs; however, relatively few have been attributed to β-cell function. Here, we will describe recent findings on those coregulators with direct roles in maintaining islet β-cell health and identity and discuss how disruption of coregulator activity is associated with diabetes pathogenesis.
  • Thumbnail Image
    Item
    Dynamic regulation of pancreatic β cell function and gene expression by the SND1 coregulator in vitro
    (Taylor & Francis, 2023) Kanojia, Sukrati; Davidson, Rebecca K.; Conley, Jason M.; Xu, Jerry; Osmulski, Meredith; Sims, Emily K.; Ren, Hongxia; Spaeth, Jason M.; Biochemistry and Molecular Biology, School of Medicine
    The pancreatic β cell synthesizes, packages, and secretes insulin in response to glucose-stimulation to maintain blood glucose homeostasis. Under diabetic conditions, a subset of β cells fail and lose expression of key transcription factors (TFs) required for insulin secretion. Among these TFs is Pancreatic and duodenal homeobox 1 (PDX1), which recruits a unique subset of transcriptional coregulators to modulate its activity. Here we describe a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), which has been shown to facilitate protein-protein interactions and transcriptional control through diverse mechanisms in a variety of tissues. PDX1:SND1 interactions were confirmed in rodent β cell lines, mouse islets, and human islets. Utilizing CRISPR-Cas9 gene editing technology, we deleted Snd1 from the mouse β cell lines, which revealed numerous differentially expressed genes linked to insulin secretion and cell proliferation, including limited expression of Glp1r. We observed Snd1 deficient β cell lines had reduced cell expansion rates, GLP1R protein levels, and limited cAMP accumulation under stimulatory conditions, and further show that acute ablation of Snd1 impaired insulin secretion in rodent and human β cell lines. Lastly, we discovered that PDX1:SND1 interactions were profoundly reduced in human β cells from donors with type 2 diabetes (T2D). These observations suggest the PDX1:SND1 complex formation is critical for controlling a subset of genes important for β cell function and is targeted in diabetes pathogenesis.
  • Thumbnail Image
    Item
    Investigating the Impact of SND1 and CHD3 ad PDX1 Interacting Partners on β Cell Function
    (2024-08) Kanojia, Sukrati; Spaeth, Jason; Molina, Carmella Evans; Linnemann, Amelia K.; Mastracci, Teresa; Elmendorf, Jeffrey S.
    Pancreatic β cells are integral in synthesizing, packaging, and secreting insulin, crucial for maintaining blood glucose homeostasis. However, in diabetic conditions, some β cells lose function of transcription factors (TFs), which drive expression of genes critical for insulin secretion. Among these, PDX1 plays a vital role in pancreas development and mature β cell function. The activity of PDX1 is modulated by coregulators like the SWI/SNF chromatin remodeling and Nucleosome Remodeling and Deacetylase (NuRD) complexes. Our study unveils a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), known for its role in facilitating protein-protein interactions and transcriptional control across tissues. Confirming PDX1:SND1 interactions in rodent and human β cells, we employed CRISPR-Cas9 to delete Snd1, revealing altered gene expression associated with insulin secretion and cell proliferation, which were confirmed through functional analyses, highlighting the importance of SND1 in β cell function. Notably, PDX1:SND1 interactions were reduced in human β cells from type 2 diabetes (T2D) donors, indicating its role in diabetes pathogenesis. Additionally, our investigation into the NuRD complex discovered interactions between PDX1 and CHD4, a helicase within the complex, are crucial for modulating PDX1 transcriptional activity in β cells. Deletion of Chd4 led to increased CHD3 protein levels, prompting us to explore the role of CHD3 in compensating for loss of CHD4. β cell specific-inducible deletion of Chd3 alone did not impact glucose homeostasis, whereas concurrent deletion of Chd3 and Chd4 resulted in severe glucose intolerance, reduced β cell mass, and compromised insulin release. Loss of both subunits led to β cell dysfunction and loss of identity, emphasizing the compensatory role of CHD3. Future investigations will evaluate gene expression and chromatin accessibility changes in Chd3/Chd4-deficient β cells, along with assessing the impact of diabetes on PDX1:CHD3 interactions in T2D human donor tissues.
  • Thumbnail Image
    Item
    A Role for MicroRNA-146a-5p Mediated Regulation of Stromal Interaction Molecule 1 and Store Operated Calcium Entry in the Pancreatic Beta-Cell in Response to Cytokine Mediated Stress
    (2019-09) Kanojia, Sukrati; Goebl, Mark; Evans Molina, Carmella; Mosley, Amber; Wek, Ronald C.
    Store-operated Ca2+ entry (SOCE) is involved in the maintenance of endoplasmic reticulum (ER) Ca2+ levels. The SOCE involves Stromal Interaction Molecule 1 (STIM1), distributed throughout the ER, and Orai1 channels, dispersed on the plasma membrane. SOCE is activated by the depletion of ER Ca2+ causing STIM1 to induce ER expansion and recruits Orai1 channels thus replenishing ER Ca2+. We reported downregulation of STIM1 in human islets from donors with type 2 diabetes (T2D) and in INS-1 β-cells treated with cytokines, and loss of STIM1 expression impairs β-cell SOCE, ER stress, and reduced insulin secretion. However, the regulatory mechanisms of STIM1 downregulation are unknown. To test this, actinomycin D and cycloheximide chase assay was performed to define whether IL-1β treatment impacted STIM1 mRNA or protein half-life. IL-1β had no impact on mRNA or protein decay. MicroRNAs (miRNAs), a class of small non-coding RNAs can regulate gene expression post-transcriptionally by binding to complementary regions in the 3’ untranslated region (UTR) of target mRNAs, affecting mRNA stability and translatability. The objective of this study was to establish miRNA regulation of STIM1 expression and altered SOCE. To identify potential miRNA candidates, RNA sequencing was done in human islets, treated with IL-1β and IFN-γ for 24 hrs. A total of 20 miRNAs were differentially expressed using a FC value of ≥ 1.5 and a p value of < 0.05. Of these, two miRNAs (miR-146a-5p and miR-4640-5p) were predicted by TargetScan to bind the 3’UTR of STIM1.To validate these findings, INS-1 β-cells, and human islets were treated with or without IL-1β. Only miR-146a-5p was upregulated in both systems. Consistent with inverse correlation, INS-1 β-cells transfected with miR-146a-5p mimic showed reduced STIM1 expression. To test whether miR-146a-5p inhibition preserves STIM1 expression, INS1 cells were treated with miR-146a-5p inhibitor along with IL-1β and inhibition of miR-146a-5p led to partial preservation of STIM1 expression. Future studies will test the effect of miR-146a-5p mimics and inhibitors on SOCE. The results indicate that the stress induced by IL-1β leads to induction of miR-146a-5p, which may then target STIM1 mRNA. Such studies could enable broader implementation of miRNA in βcell dysfunction.
  • Thumbnail Image
    Item
    The Chd4 Helicase Regulates Chromatin Accessibility and Gene Expression Critical for β-Cell Function In Vivo
    (American Diabetes Association, 2023) Davidson, Rebecca K.; Kanojia, Sukrati; Wu, Wenting; Kono, Tatsuyoshi; Xu, Jerry; Osmulski, Meredith; Bone, Robert N.; Casey, Nolan; Evans-Molina, Carmella; Sims, Emily K.; Spaeth, Jason M.; Biochemistry and Molecular Biology, School of Medicine
    The transcriptional activity of Pdx1 is modulated by a diverse array of coregulatory factors that govern chromatin accessibility, histone modifications, and nucleosome distribution. We previously identified the Chd4 subunit of the nucleosome remodeling and deacetylase complex as a Pdx1-interacting factor. To identify how loss of Chd4 impacts glucose homeostasis and gene expression programs in β-cells in vivo, we generated an inducible β-cell-specific Chd4 knockout mouse model. Removal of Chd4 from mature islet β-cells rendered mutant animals glucose intolerant, in part due to defects in insulin secretion. We observed an increased ratio of immature-to-mature insulin granules in Chd4-deficient β-cells that correlated with elevated levels of proinsulin both within isolated islets and from plasma following glucose stimulation in vivo. RNA sequencing and assay for transposase-accessible chromatin with sequencing showed that lineage-labeled Chd4-deficient β-cells have alterations in chromatin accessibility and altered expression of genes critical for β-cell function, including MafA, Slc2a2, Chga, and Chgb. Knockdown of CHD4 from a human β-cell line revealed similar defects in insulin secretion and alterations in several β-cell-enriched gene targets. These results illustrate how critical Chd4 activities are in controlling genes essential for maintaining β-cell function. Article highlights: Pdx1-Chd4 interactions were previously shown to be compromised in β-cells from human donors with type 2 diabetes. β-Cell-specific removal of Chd4 impairs insulin secretion and leads to glucose intolerance in mice. Expression of key β-cell functional genes and chromatin accessibility are compromised in Chd4-deficient β-cells. Chromatin remodeling activities enacted by Chd4 are essential for β-cell function under normal physiological conditions.