Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations.

Robertson, R. Paul; Zhang, Hui Jian; Pyzdrowski, Kathryn L.; Walseth, Timothy F.

doi:10.1172/jci115865

Cited by 217 publications

(160 citation statements)

References 49 publications

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“…In fact, we have extended our studies toward examination of the expression of C/EBP␤ in pancreatic ␤-cells in animal models of diabetes mellitus, with preliminary results implying an involvement of this factor in the pathophysiology of glucotoxic alterations during the development and progression of this disease. 5 …”

Section: Discussionmentioning

confidence: 99%

“…Studies using in vivo animal models and in vitro ␤-cell lines have demonstrated that a reduction of insulin gene transcription by glucose toxicity is associated with the loss of transactivator proteins such as IDX-1/IPF-1/STF-1 and RIPE3b1-binding protein (5)(6)(7)(8)(9)(10). Because insulin gene transcription is both positively and negatively regulated, we sought to identify repressors that might also mediate the effects of glucose toxicity on insulin gene transcription.…”

mentioning

confidence: 99%

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Pancreatic β-Cell-specific Repression of Insulin Gene Transcription by CCAAT/Enhancer-binding Protein β

Lu¹,

Seufert²,

Habener

1997

Journal of Biological Chemistry

120

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Chronic exposure of ␤-cells to supraphysiologic glucose concentrations results in decreased insulin gene transcription. Here we identify the basic leucine zipper transcription factor, CCAAT/enhancer-binding protein ␤ (C/EBP␤), as a repressor of insulin gene transcription in conditions of supraphysiological glucose levels. C/EBP␤ is expressed in primary rat islets. Moreover, after exposure to high glucose concentrations the ␤-cell lines HIT-T15 and INS-1 express increased levels of C/ EBP␤. The rat insulin I gene promoter contains a consensus binding motif for C/EBP␤ (CEB box) that binds C/EBP␤. In non-␤-cells C/EBP␤ stimulates the activity of the rat insulin I gene promoter through the CEB box. Paradoxically, in ␤-cells C/EBP␤ inhibits transcription, directed by the promoter of the rat insulin I gene by direct protein-protein interaction with a heptad leucine repeat sequence within activation domain 2 of the basic helix-loop-helix transcription factor E47. This interaction leads to the inhibition of both dimerization and DNA binding of E47 to the E-elements of the insulin promoter, thereby reducing functionally the transactivation potential of E47 on insulin gene transcription. We suggest that the induction of C/EBP␤ in pancreatic ␤-cells by chronically elevated glucose levels may contribute to the impaired insulin secretion in severe type II diabetes mellitus.Insulin is a hormone essential for the control of mammalian glucose homeostasis and is produced predominantly in pancreatic ␤-cells of adult animals (1). The expression of the insulin gene occurs to a large extent at the level of transcription. Control elements residing in the 5Ј 350-base pair sequence flanking exon 1 of the rat insulin I gene are sufficient to direct ␤-cell-specific expression (2) (Fig. 1A). Arrays of A and E elements 1 (Far-FLAT, Nir-P1) constitute symmetrical enhansons that cooperatively account for Ͼ90% of the transcriptional activity of the insulin gene promoter (3). The E elements are recognition motifs for transcription factors in the basic helixloop-helix (bHLH) 2 family, such as E12 and E47, which activate the insulin promoter in close synergism with A element binding homeobox transcription factors, such as IDX-1.Chronic hyperglycemia may contribute to the pancreatic ␤-cell dysfunction observed in patients with type II diabetes, a phenomenon attributed to the concept of glucose toxicity (4). Studies using in vivo animal models and in vitro ␤-cell lines have demonstrated that a reduction of insulin gene transcription by glucose toxicity is associated with the loss of transactivator proteins such as IDX-1/IPF-1/STF-1 and RIPE3b1-binding protein (5-10). Because insulin gene transcription is both positively and negatively regulated, we sought to identify repressors that might also mediate the effects of glucose toxicity on insulin gene transcription. In this report we describe CCAAT/enhancer binding protein-␤ (C/EBP␤) as a glucoseinduced repressor of insulin gene transcription.C/EBPs are a family of transcription factors that regulate...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Pancreatic β-Cell-specific Repression of Insulin Gene Transcription by CCAAT/Enhancer-binding Protein β

Lu¹,

Seufert²,

Habener

1997

Journal of Biological Chemistry

120

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“…The reversal of diabetes in NOD mice by CFA and repeated exposure to C57 splenocytes indicated that restoration of endogenous islet function is achievable without islet transplantation and despite the poor glycemic control attained by insulin injections. The beneficial influence of glycemic control on the growth, survival, and function of cultured islets, as well as of transplanted islets in nonautoimmune settings, has been demonstrated (35,36). To determine whether the restoration of endogenous β cell function could be achieved more consistently with better control of blood glucose, we replaced insulin injections with the intraperitoneal implantation of alginate-encapsulated C57 mouse islets.…”

Section: Figurementioning

confidence: 99%

Reversal of established autoimmune diabetes by restoration of endogenous β cell function

Ryu¹,

Kodama²,

Ryu³

et al. 2001

J. Clin. Invest.

163

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“…Glucose toxicity in the pancreatic islet beta cell secondarily leads to further defects in beta cell function, including decreases in insulin reporter activity, gene expression, content, and secretion (3). Antioxidants have been shown to prevent these adverse changes in experimental models (4,5).…”

mentioning

confidence: 99%

Oxidative Stress-mediated, Post-translational Loss of MafA Protein as a Contributing Mechanism to Loss of Insulin Gene Expression in Glucotoxic Beta Cells

Harmon

Stein

Robertson

2005

Journal of Biological Chemistry

213

166

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After the onset of type 2 diabetes, chronic hyperglycemia causes glucotoxic changes in many tissues (1, 2). Glucose toxicity in the pancreatic islet beta cell secondarily leads to further defects in beta cell function, including decreases in insulin reporter activity, gene expression, content, and secretion (3). Antioxidants have been shown to prevent these adverse changes in experimental models (4, 5). We previously observed that loss of insulin gene expression is accompanied by decreased binding to the promoter region of two important transcription factors, PDX-1 1 (STF-1, IDX-1, IPF-1) and RIPE-3b1 activator (6 -8). PDX-1 is required for pancreatic development and is a key regulator of insulin gene expression. Mutations within the RIPE-3b1/C1 element of the insulin promoter markedly reduce glucose-responsive insulin gene expression (7). We also reported that loss of RIPE-3b1 binding precedes the loss of PDX-1 binding to the insulin promoter as glucotoxicity develops (8). The chronology of these losses is likely to be important in light of a recent report that RIPE-3b1/MafA directly activates PDX-1 transcription (9).The work in this study features the use of HIT-T15 cells, a glucose-responsive beta cell line that has proven over the past decade to reproduce the molecular changes in gene expression that are caused by glucose toxicity in vivo in animal models. Since isolated islets cannot be chronically cultured to study the adverse effects of high glucose concentrations over many months, this cell line is a valuable surrogate. In our previous work, reconstitution of late passage glucotoxic HIT-T15 cells by transient transfection with PDX-1 partially restored insulin promoter activity (8). Since RIPE-3b1 had not yet been cloned, we were unable to extend our studies with this transcription factor but hypothesized that reconstitution of the cells with both PDX-1 and RIPE-3b1 activator would lead to greater recovery of insulin promoter activity. With the recent cloning and identification of RIPE-3b1 activator as MafA (10 -12), we have been able to perform new studies to examine 1) whether levels of MafA mRNA and protein are decreased in glucotoxic beta cells; 2) the mechanism of MafA protein degradation; 3) whether the antioxidant, N-acetylcysteine, can prevent glucotoxicity-induced loss of MafA protein and binding to the insulin promoter; and 4) whether overexpression of MafA and PDX-1 together can restore insulin promoter activity and mRNA levels more fully than PDX-1 alone. MATERIALS AND METHODSCell Culture-HIT-T15 cells were maintained in RPMI 1640 media containing 10% fetal bovine serum and 11.1 mM glucose as described previously (13). Cells were categorized as early passage (p71-75) or late passage (p123-128), with each passage occurring weekly. Chronic culturing of HIT-T15 cells with N-acetyl-L-cysteine (Sigma; 0.5, 1, or 5 mM) added to the media was begun at p70.

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Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations.

Cited by 217 publications

References 49 publications

Pancreatic β-Cell-specific Repression of Insulin Gene Transcription by CCAAT/Enhancer-binding Protein β

Pancreatic β-Cell-specific Repression of Insulin Gene Transcription by CCAAT/Enhancer-binding Protein β

Reversal of established autoimmune diabetes by restoration of endogenous β cell function

Oxidative Stress-mediated, Post-translational Loss of MafA Protein as a Contributing Mechanism to Loss of Insulin Gene Expression in Glucotoxic Beta Cells

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