Volume 117, Issue 1, Pages 132-139 (July 1999) The glucose-6 phosphatase gene is expressed in human and rat small intestine: Regulation of expression in fasted and diabetic rats  Fabienne Rajas, Nathalie Bruni, Sandrine Montano, Carine Zitoun, Gilles Mithieux  Gastroenterology  Volume 117, Issue 1, Pages 132-139 (July 1999) DOI: 10.1016/S0016-5085(99)70559-7 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Tissue specificity of Glc6Pase gene expression in rats and humans. (A) Amplification of rat Glc6Pase cDNA (1095 bp) by RT-PCR from 1 μg of total RNA from all tissues in fed rats. From 100 μL of amplification medium (see Materials and Methods), 20 μL was loaded on a 1.5% agarose gel in all lanes. On the left lane, the digestion products of phage (lambda) by EcoRI/HindIII were analyzed (DNA fragments appearing were 2027, 1904, 1584, 1330, 983, and 831 bp long, from top to bottom). (B) Amplification of human Glc6Pase cDNA (1079 bp) by RT-PCR from 0.2 μg of total RNA from all tissues. From 50 μL of amplification medium, 20 μL was loaded. (C) Comparative analysis of abundance of Glc6Pase mRNA in various tissues from fed rats by Northern blotting. Fourteen micrograms of total RNA (based on UV quantification) was electrophoresed and blotted in each track. Autoradiographs were scanned, and Glc6Pase mRNA was quantified using a Vernon densitometer. The loading conditions were normalized via densitometric quantification of the 28S RNAs visualized using ethidium bromide in the agarose gels just before transfer on nylon membranes (<10% of variation among tracks was observed in all cases). It was also determined after the transfer that no RNA remained in the agarose gel. The arrowheads on the left indicate the position of the 28S (up) and 18S (down) RNAs. BAT, brown adipose tissue; UB, urinary bladder; WAT, white adipose tissue. Gastroenterology 1999 117, 132-139DOI: (10.1016/S0016-5085(99)70559-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of differentiation on the expression of Glc6Pase gene in Caco-2 cells. Amplification of human Glc6Pase cDNA (1079 bp) from 1 μg of total RNA from the indicated human tissues and cell lines. Undifferentiated (undif) Caco-2 cells were cultured for 2 days after confluence and did not show any cell domes. Differentiated (dif) Caco-2 cells were cultured for 9 days after confluence and showed numerous cell domes. From 50 μL of amplification medium, 15 μL was loaded on a 1.5% agarose gel. On the left and right lanes, molecular mass markers were loaded on the gel. DNA fragments appearing were 2000, 1500, 1000, and 800 bp long from top to bottom. Gastroenterology 1999 117, 132-139DOI: (10.1016/S0016-5085(99)70559-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of diabetes and insulin treatment on Glc6Pase mRNA abundance and enzymatic activity in rat small intestine. (A and B) Northern blot analysis of Glc6Pase mRNA abundance in normal fed (N), diabetic (D), and insulin-treated diabetic (DT) rats. (C and D) Densitometric analysis of Northern blots obtained from 4 animals in each group. The Glc6Pase mRNA abundances were referred to a standard Glc6Pase mRNA chosen arbitrarily (from the duodenum of a normal fed rat), which was analyzed in parallel with other samples in all blots analyzed in this report. The mean value for the duodenum from normal fed rats was 100%, and all other values were corrected accordingly. Therefore, all results presented in Figures 3 and 4 are expressed as percentages of the mean value for the duodenum from normal fed rats and thus may be compared quantitatively. (E and F) Specific Glc6Pase activity assayed in tissue homogenates in the presence of 20 mmol/L Glc6P under the conditions described in Materials and Methods based on β-glycerophosphatase activity subtraction. Results are expressed as means ± SEM (n = 4). *P < 0.05, **P < 0.01, significantly different from N value; oP < 0.05, ooP < 0.01, significantly different from D value. Gastroenterology 1999 117, 132-139DOI: (10.1016/S0016-5085(99)70559-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of fasting and refeeding on Glc6Pase mRNA abundance and enzymatic activity in rat small intestine. (A–C) Northern blot analysis of Glc6Pase mRNA abundance in normal fed (N), 48-hour-fasted (F), and refed (R) rats. (D–F) Densitometric analysis of Northern blots obtained from 5 animals in each group (refer to Figure 2 for quantitative analysis). (G–I) Specific Glc6Pase activity assayed in tissue homogenates as described in Figure 3. Results are expressed as means ± SEM (n = 5). *P < 0.05, **P < 0.01, significantly different from N value; oP < 0.05, ooP < 0.01, significantly different from F value. Gastroenterology 1999 117, 132-139DOI: (10.1016/S0016-5085(99)70559-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions