Impaired Hepatocyte Glucose Transport Protein (GLUT2) Internalization in Chronic Pancreatitis

Abstract

Chronic pancreatitis (CP) is associated with impaired glucose tolerance and with reduced hepatic sensitivity to insulin. We have previously shown that in normal and sham-operated rats, insulin suppresses hepatic glucose production, and this suppression is associated with a decrease in the hepatocyte plasma membrane-bound quantity of the facilitative glucose transport protein GLUT2. The insulin-mediated reduction in membrane-bound GLUT2 is impaired in CP, and may play a role in the glucose intolerance associated with CP. To determine whether GLUT2 is actively internalized and whether this mechanism is disordered in CP, livers from fed and fasting rats in whom CP had been induced 2-3 months earlier by pancreatic duct oleic acid infusion, and in sham-operated (sham) rats, were fractionated to yield endosome (E)- and plasma membrane (PM)-enriched fractions. Forty-five minutes after duodenal intubation alone (fasting) or intubation plus duodenal feeding, livers were removed, homogenized and ultracentrifuged, and microsomal pellets were separated by sucrose density gradient ultracentrifugation. GLUT2 content of fractions was determined by Western blotting and scanning densitometry. The E:PM ratio of GLUT2 increased from 0.68 +/- 0.11 (mean +/- SEM) in fasting sham livers (n = 8) to 1.04 +/- 0.09 in fed sham livers (n = 8; p < 0.05). However, there was no change in the E:PM ratio of GLUT2 in CP livers after duodenal feeding (0.90 +/- 0.12 vs. 0.86 +/- 0.10; n = 8,8; p = NS). To test our findings using confocal laser scanning microscopy, liver specimens from fed and fasting CP and sham rats were minced, fixed in 4% paraformaldehyde, sectioned, and stained with rabbit antirat GLUT2 antibody followed by rhodamine-labeled secondary antibody. GLUT2 was quantified by mean pixel intensity in an 8 x 16-pixel area of PM and a 16 x 16-pixel area of cytosol (CYT) in each of 30 random cells/field (400x) in each of three rats per group. As in the fractionation study, duodenal feeding increased the CYT:PM ratio of GLUT2 from 0.75 +/- 0.01 in fasting sham liver to 0.86 +/- 0.01 in fed sham liver (p < 0.0001), while the CYT:PM ratio in CP remained unchanged. We conclude that feeding induces a shift in GLUT2 from the plasma membrane to the endosomal pool. The feeding-induced internalization of GLUT2 is absent in livers from rats with CP and may play a role in the glucose intolerance associated with CP.

Insulin Receptor (IR) and Glucose Transporter 2 (GLUT2) Proteins Form a Complex on the Rat Hepatocyte Membrane

“…Similarly, the mean ratio of endosome:plasma membrane GLUT2 was 0.68±0.11 in fasted rats. The mean ratio of endosome:plasma membrane GLUT2, as previously shown [12], increased to 1.04±0.09 after feeding (p<0.02). Figure 2 shows a simultaneous graph of IR endosome:plasma membrane content vs. GLUT2 endosome:plasma membrane content.…”

“…It is interesting to note that not all of the GLUT2 is internalized in the normal hepatic response to the post-prandial state. Our previous data showed that after an intraduodenal feeding bolus, which stimulates the release of insulin and the suppression of hepatic glucose production, a substantial fraction (15-53%) of GLUT2 endocytosis occurs [12]. A prior study showed that this same effect was seen during in vitro liver perfusion in response to graduated doses of insulin [11].…”

“…Insulin perfused livers, however, show a marked decrease in the presence of GLUT2 in the cell membrane fraction which is dose-dependent, and parallels the insulin-mediated reduction in hepatic glucose production [11]. Subsequent studies in our laboratory have shown this disappearance to be mediated by an endocytotic process [12].…”

Pancreatogenic Diabetes: Special Considerations for Management

“…Subsequently, this group reported that hepatocyte IR gene expression was regulated, in part, by PP [43]. In addition to impaired hepatic IR expression, other studies revealed that the internalization of hepatocyte IR and the principal glucose transporter protein GLUT2 is impaired in CP [44,45]. This defect in clathrin-mediated endocytosis of the putative IR-GLUT2 complex [46] was not altered by PP replacement, and likely represents a second defect in IR function in pancreatogenic diabetes [29].…”

Antihyperglycemic and Antioxidant Properties of Caffeic Acid in db/db Mice

“…The livers and adipose tissues were prepared according to Nathan et al (2001) with a slight modification. In brief, the tissue was homogenized with a buffer containing 250 mM sucrose, pH 7.4, 10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine hydrochloride hydrate, and 1 mM dithiothreitol.…”