The loss of GLUT2 expression in the pancreatic β-cells of diabetic db/db mice is associated with an impaired DNA-binding activity of islet-specific trans-acting factors

Abstract

GLUT2 expression is reduced in the pancreatic β-cells of several diabetic animals. The transcriptional control of the gene in β-cells involves at least two islet-specific DNA-binding proteins, GTIIa and PDX-1, which also transactivates the insulin, somatostatin and glucokinase genes. In this report, we assessed the DNA-binding activities of GTIIa and PDX-1 to their respective cis-elements of the GLUT2 promoter using nuclear extracts prepared from pancreatic islets of 12 week old db/db diabetic mice. We show that the decreased GLUT2 mRNA expression correlates with a decrease of the GTIIa DNA-binding activity, whereas the PDX-1 binding activity is increased. In these diabetic animals, insulin mRNA expression remains normal. The adjunction of dexamethasone to isolated pancreatic islets, a treatment previously shown to decrease PDX-1 expression in the insulin-secreting HIT-T15 cells, has no effect on the GTIIa and PDX-1 DNA-binding activities. These data suggest that the decreased activity of GTIIa, in contrast to PDX-1, may be a major initial step in the development of the β-cell dysfunction in this model of diabetes.

Introduction

Non-insulin-dependent diabetes mellitus (NIDDM) is believed to occur in individuals with insulin resistance coupled with abnormal glucose-stimulated insulin secretion by pancreatic β-cells (Polonsky et al., 1996). Glucose-induced insulin secretion occurs through a signalling pathway which requires glucose metabolism, a process which is initiated by the uptake of glucose by the glucose transporter GLUT2 (Thorens et al., 1988, Orci et al., 1989). In animals with diabetes, the impaired insulin secretory response is correlated with a reduced expression of GLUT2, which represents an early biochemical marker of the disease (Johnson et al., 1990, Orci et al., 1990a, Orci et al., 1990b, Thorens et al., 1990, Thorens et al., 1992, Ohneda et al., 1993). No decrease of GLUT2 abundance has been reported in the liver or the kidney of the diabetic animals (Thorens et al., 1992), which suggests that expression of the GLUT2 gene is under the control of different factors in pancreatic β-cells compared to other GLUT2-expressing tissues. These factors are sensitive to the action of circulating molecules present in the diabetic environment which are distinct from glucose or insulin (Thorens et al., 1992). Furthermore, Tokuyama et al. (1995)have described abnormal expression of several genes beside GLUT2 within the pancreatic islet of the diabetic Zucker rat. Taken together, these experiments suggest that unidentified circulating factors present in the diabetic environment alter the overall expression of many genes in the pancreatic β-cells, possibly by altering the activity of a restricted number of islet-specific transcription factors.

The β-cell specific expression of the murine GLUT2 gene involves at least two distinct β-cell specific transcription factors: the GTIIa complex and the homeodomain containing transcription factor PDX-1 (known as IDX-1, STF-1 or IPF1), which also controls expression of the insulin, somatostatin and glucokinase genes (Leonard et al., 1993, Ohlsson et al., 1993, Miller et al., 1994, Bonny et al., 1995, Waeber et al., 1996, Watada et al., 1996). Expression of PDX-1 is regulated by glucose, fatty acids and by glucocorticoids (Olson et al., 1993, MacFarlane et al., 1994, Olson et al., 1995, Sharma et al., 1995, Gremlich et al., 1997a, Sharma et al., 1997, Zangen et al., 1997). Exposure of pancreatic islets to high glucose results in a rapid phosphorylation of the protein and an increased DNA-binding activity to its target sequences of the insulin promoter (MacFarlane et al., 1994). However, long-term exposure of insulin-secreting HIT-T15 cells to high glucose concentrations leads to a reduced content of the PDX-1 transcription factor and to a concomitant decreased expression of the insulin gene (Olson et al., 1993, Olson et al., 1995, Sharma et al., 1995). In vivo, hyperglycaemia induced by 95% pancreatectomy leads to a decreased expression of the PDX-1 mRNA (Zangen et al., 1997). Dexamethasone also induces a rapid decrease in PDX-1 expression in HIT-T15 cells, which is correlated by a 4–5-fold decrease in insulin mRNA content, whereas fatty acids induce a decreased PDX-1 DNA-binding activity and mRNA expression in isolated pancreatic islets (Gremlich et al., 1997a, Sharma et al., 1997). These observations raise the possibility that alteration of PDX-1 abundance and/or function may result from metabolic disturbances present in the diabetic state such as hyperglycaemia, high circulating free fatty acids and/or high glucocorticoids. This in turn may lead to abnormal expression of target genes such as GLUT2, insulin or glucokinase and subsequently to an abnormal secretory response of the pancreatic β-cells.

The GTIIa complex was characterized as a β-cell specific DNA-binding activity associated with the proximal GLUT2 promoter sequences (Bonny et al., 1995). This region of the promoter does not interact with PDX-1 and is able to confer islet-specific expression both in vivo, in a transgenic mouse approach and, in vitro, in cultured cell lines (Bonny et al., 1995, Waeber et al., 1995). In vitro, the abundance of the GTIIa complex parallels closely the level of endogenous GLUT2 mRNA: GTIIa is highly expressed in INS-1 where GLUT2 is abundantly transcribed, but is expressed to much lower extent in βTC-3 and in RIN 1027-B2 cells where endogenous GLUT2 expression is also lower (Bonny et al., 1995). No GTIIa binding activity is observed in non-pancreatic cell lines (Bonny et al., 1995).

In this report, we analysed the DNA-binding activities of the β-cell-specific transcription factors which control GLUT2 gene expression in pancreatic islets of normal (db/+) and diabetic (db/db) mice. Our results show that the decreased GLUT2 gene expression in db/db mice occurs in the absence of a decreased DNA-binding activity of PDX-1. However, a decrease of the GTIIa activity was observed and may represent an important element in the control of the decreased GLUT2 expression in the pancreatic islets of this animal model of diabetes.