Evolutionary structural and functional conservation of an ortholog of the GLUT2 glucose transporter gene (SLC2A2) in zebrafish

In mammals, GLUT2 plays an essential role in glucose homeostasis. From an evolutionary perspective, relatively little is known about the biology of GLUT2, or other GLUTs, in nonmammalian vertebrates. Here, we have conducted studies to functionally characterize GLUT2 in zebrafish. First, we cloned the zebrafish ortholog of GLUT2 (zfGLUT2) encoding a protein of 504 amino acids with high-sequence identity to other known vertebrate GLUT2 proteins. The zfGLUT2 gene consists of 11 exons and 10 introns, spanning 20 kb and mapping to a region of chromosome 2 that exhibits conserved synteny with human chromosome 3. When expressed in Xenopus oocytes, zfGLUT2 transported 2-deoxyglucose (2-DG) with similar affinity than mammalian GLUT2 ( Km of 11 mM). Transport of 2-DG was competed mostly by d-fructose and d-mannose and was inhibited by cytochalasin B. During early development, zfGLUT2 expression was detected already at 10 h postfertilization and remained elevated in 5-day larvae, when it was clearly localized to the liver and intestinal bulb. In the adult, zfGLUT2 expression was highest in testis, brain, skin, kidney, and intestine, followed by liver and muscle. In the intestine, zfGLUT2 transcripts were detected in absorptive enterocytes, and its mRNA levels were altered by fasting and refeeding, suggesting that its expression in the intestine may be regulated by the nutritional status. These results indicate that the structure and function of GLUT2 has been remarkably well conserved during vertebrate evolution and open the way for the use of zebrafish as a model species in which to study the biology and pathophysiology of GLUT2.

CCAAT/enhancer binding protein regulates the promoter activity of the rat GLUT2 glucose transporter gene in liver cells

The liver-specific expression of the GLUT2 glucose transporter gene is suppressed in cultured hepatoma cell lines as well as in hepatocytes in primary culture. To understand the underlying mechanism involved in this process, we analysed the rat GLUT2 promoter region. A DNase I footprinting assay with rat liver nuclear extract revealed eight protected regions within a -500 bp region of the GLUT2 promoter (sites A to H). Three of these sites (B, F and H) were occupied by transcription factors that are considerably enriched in liver cells compared with spleen or kidney. The proteins binding to these sites were investigated by a combination of DNase I footprinting assay and electrophoretic mobility-shift assay with the addition of specific oligonucleotide competitors and specific antibody against known transcription factors. As a result it was revealed that hepatocyte nuclear factor 3 binds to site B (-120 to -70), and CCAAT/enhancer binding protein α (C/EBPα) and C/EBPβ bind to site F (-375 to -356) and site H (-500 to -471). The binding of C/EBP to sites F and H was markedly decreased within 4 h when liver cells were subjected to primary culture, suggesting that C/EBP might be responsible for the decreased expression of GLUT2 in this process. In contrast, Western blot analysis revealed that C/EBPα began to decrease after 1 h of hepatocyte culture, and C/EBPβ was not changed significantly throughout the culture period, suggesting that C/EBP could be regulated at the transcriptional level as well as the post-translational level when hepatocytes were put in culture. To confirm the role of C/EBP in the regulation of GLUT2 promoter activity, sites F and H were ligated to a chloramphenicol acetyltransferase (CAT) reporter gene and co-transfected with a C/EBP expression vector into HepG2 cells. The co-expression of C/EBPα and C/EBPβ resulted in 9.1-fold and 3.8-fold increases of CAT activities in the site F-CAT and site H-CAT constructs respectively. These results indicate that C/EBPα and C/EBPβ regulate the promoter activity of the GLUT2 gene and might be responsible for the down-regulation of the GLUT2 gene when hepatocytes are subjected to primary culture.

Effects of sodium butyrate on glucose transporter and glucose-phosphorylating enzyme gene expression in RINm5F insulinoma cells

ABSTRACT RINm5F insulinoma cells show a defective physiological insulin secretory response to glucose stimulation. The short chain carbonic acid sodium butyrate induced a growth arrest during a 72-h tissue culture period. In contrast to control RINm5F cells, 2 mm glucose increased insulin secretion by more than 70% in these sodium butyrate-treated cells (1 mm) without any further increase of the secretory rate between 2 and 20 mm glucose. This effect of sodium butyrate on insulin secretion was assessed in comparison with its effect on gene expression of the GLUT1 and GLUT2 glucose transporter, hexokinase type I and type II, glucokinase and insulin. Sodium butyrate at a 1 mm concentration decreased GLUT1 gene expression by nearly 50%, but did not induce gene expression of the low-affinity GLUT2 glucose transporter above the detection limit. Furthermore, sodium butyrate increased glucokinase gene expression by more than 50% and hexokinase type II gene expression by more than 100%, while insulin gene expression was increased only by 24%. Hexokinase type II enzyme activity was increased by more than 100% without a concomitant significant change of the glucokinase enzyme activity. Sodium butyrate (2 mm) caused effects comparable with those of 1 mm sodium butyrate. Thus the improved insulin secretory responsiveness of RINm5F insulinoma cells after sodium butyrate treatment at low non-physiological millimolar glucose concentrations can be interpreted as a result of an increased hexokinase-mediated metabolic flux rate through the glycolytic chain.